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Patient-derived xenograft models of human lymphomas for experimental immunotherapy
Jakša, Radek ; Klener, Pavel (advisor) ; Škarda, Jozef (referee) ; Kalinová, Markéta (referee)
Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDX) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDX keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We derived 15 PDX models from patients with various subtypes of aggressive lymphomas. We implemented complex genetic and immunohistochemical analysis of the established PDX models head-to-head with the patient's primary lymphoma cells, from which the PDXs were derived. We clearly confirmed that the established PDX cells shared majority of somatic mutations with the patient's primary cells, from which they were derived. Thus, from the genetic perspective the PDX models represent relevant tools for the study of lymphoma biology. Immunohistochemistry analysis of selected antigens revealed some differences between the PDXs and patients' primary cells. Importantly, the analysis demonstrated complete loss of non-malignant cellular components of the tumor microenvironment frequently observed in lymphoma infiltrated lymph nodes,...
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In vivo application of holographic endoscopy
Tučková, Tereza ; Brzobohatý,, Oto (referee) ; Bouchal, Petr (referee) ; Uhlířová, Hana (advisor)
Pokrok v porozumění komplexním mozkovým funkcím závisí na schopnosti opticky dosáhnout jakékoli vybrané struktury a oblasti živého mozku se subbuněčným rozlišením při minimálním poškození tkáně. Zpřístupňování hlubších oblastí tkání rozptylujících světlo je v současnosti umožněno zejména vývojem optických endoskopických sond, například mikroendoskopy s gradientními čočkami (GRIN) a svazky optických vláken. Pokrok v metodách holografické modulace světla dosažený v poslední době přinesl jako další nadějný směr pro zobrazování s vysokým rozlišením hluboko ve tkáních použití vícevidových optických vláken (MMF) jako zobrazovacích prvků. Ve srovnání s endoskopy založenými na GRIN čočkách a svazcích optických vláken poskytují MMF nejvyšší poměr rozlišení obrazu ku tloušťce sondy a způsobují minimální poškození tkáně. Úvodní část práce poskytuje přehled o nejmodernějších technologiích hloubkového zobrazování mozku in vivo, vícevidové vláknové endoskopii a jejích principech s cílem představit související technologii. Hlavním technologickým zaměřením práce je použití digitálního mikrozrcátkového zařízení (DMD) k modulaci světla, šířící se MMF sondou. To umožňuje rychlé rastrování fluorescenčního vzorku v zobrazovací rovině za distální hranou vlákna. Byla sestrojena optická sestava využívající tohoto principu, bylo dosaženo vysoké stability a byly pečlivě vyhodnoceny zobrazovací vlastnosti. Ty byly demonstrovány na 2D a 3D fluorescenčních fantomových vzorcích. Dále jsme vyvinuli metodu zpracování obrazu, zlepšující jeho kvalitu a umožňující dosáhnout plného potenciálu difrakčně omezeného rozlišení. Použití algoritmů využívajících regularizované iterativní inverze, případně regularizované přímé pseudoinverze, zvyšuje kontrast a rozlišení obrazu. Další cestou k ex vivo a in vivo zobrazování bylo použití geneticky modifikované myši. Identifikovali jsme vhodné myší modely a ex vivo zobrazování mozku ukázalo, že snímky trpí silným fluorescenčním signálem pozadí z oblastí mimo ohniskovou rovinu. Proto se další práce zaměřila na vývoj technologie útlumu světla založené na konfokálním principu. Byla sestrojena optická sestava pro konfokální filtraci "dírkovou clonkou" s použitím speciální sondy složené z MMF s odstupňovaným indexem lomu spojeného s MMF se skokovým indexem, a druhého DMD. Během zobrazování byl fluorescenční signál shromážděný GRIN-SI-MMF sondou filtrován ve vzdáleném poli sondy, kde se pro každý skenovací ohniskový bod vytváří prstenec. Prstencovitý signál se pak oddělí pomocí masky na DMD2, čímž se také oddělí signál pocházející z ohniska od signálu vznikajícího mimo ohnisko. Na experimentech s použitím fantomového vzorku fluorescenčních mikrokuliček i fixované mozkové tkáně bylo prokázáno, že toto konfokální filtrování vede k zeslabení signálu pozadí, tedy signálu z mimoohniskových rovin, čímž se zvyšuje kontrast a rozlišení snímků. Tento princip konfokální filtrace v holografickém endoskopu byl rovněž demonstrován pomocí nové MMF sondy s bočním zobrazováním. Práce ukazuje jen kousek skládačky dlouhodobého komplexní vývoje optimálního nástroje pro hloubkové tkáňové zobrazování s vysokým rozlišením. Holografický endoskop využívající MMF byl zdokonalen tak, že může sloužit k rutinnímu několikahodinovému zobrazování biologických tkání s možností útlumu světla pocházejícího mimo ohniskovou rovinu. Endoskop byl testován při zobrazování fantomových vzorků i fixovaných plátků myšího mozku a in vivo cév až do hloubky 5 mm.
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Neuroprotective effects of novel anorexigenic analogs of prolactin-releasing peptide (PrRP) in models of neurodegeneration in vitro and in vivo
Mengr, Anna ; Maletínská, Lenka (advisor) ; Sumová, Alena (referee) ; Hampl, Aleš (referee)
Alzheimer's disease (AD) is a progressive brain disorder characterized by extracellular beta amyloid (Aβ) plaques, intracellular neurofibrillary tangles formed by hyperphosphorylated Tau protein and neuroinflammation. Since obesity and type 2 diabetes mellitus (T2DM) have been established as risk factors for the development of neurological disorders, anorexigenic and antidiabetic peptides, such as prolactin-releasing peptide (PrRP) seem to be potential neuroprotective agents. In the first part of the study, the molecular mechanisms of action of natural PrRP31 and its lipidized analog palm11 -PrRP31 was studied in the human neuroblastoma cell line SH-SY5Y. Both compounds significantly activated the signaling pathways typical for insulin promoting cell survival and growth. Moreover, PrRP31 and palm11 -PrRP31 increased cell viability and suppressed apoptosis in methylglyoxal-stressed SH-SY5Y cells. The second part of the thesis was focused on the neuroprotective and anti-inflammatory effects of 2-month-long subcutaneous administration of palm11 -PrRP31 in the brains of APP/PS1 mice, model of Aβ pathology. Palm11 -PrRP31 significantly reduced the Aβ plaque load and microgliosis in the hippocampi, cortices, and cerebella. Furthermore, palm11 -PrRP31 increased the synaptogenesis and attenuated...
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SWI2/SNF2 ATPases with a focus on the ISWI subfamily: protein complexes and mouse models for their study
Turková, Tereza ; Stopka, Tomáš (advisor) ; Janoštiak, Radoslav (referee)
In the nucleus the DNA is packed along with proteins into a dynamic structure called chromatin. During cell cycle the chromatin structure becomes a subject to various changes. During interphase chromatin structure becomes loose while shortly before cell division it undertakes the form of highly condensed mitotic chromosomes. Structure of chromatin influences significantly mode of gene expression and its pattern. DNA-binding proteins interacting within chromatin are also necessary during this process. To gain the access to the DNA binding factors, the chromatin has to be in a loosened form. As long as the structure of the chromatin is more condensed it creates a barrier for the DNA binding proteins. Therefore it becomes obvious that the remodeling of the chromatin structure is one of the important regulators of gene expression and that the enzymes, which execute remodeling, are of great importance. One of them is ATPase Smarca5, which belongs to the protein subfamily ISWI and which creates the catalytic subunit for several different ATP-dependent chromatin remodeling complexes. Mutations of members of those complexes disturb regulation of transcription and cellular differentiation. In some cases the incorrect function of these complexes can lead to cellular transformation into a tumours state. This...
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Adipose tissue metabolism and genetically modified murine models
Irodenko, Ilariia ; Bardová, Kristina (advisor) ; Železná, Blanka (referee)
Adipose tissue plays an important role in energy and glucose homeostasis. Adipose tissue metabolism includes lipolysis and lipogenesis processes which control lipid mobilization, storage and distribution in the body. In addition to that adipose tissue is recognized as an endocrine organ which generates cytokines and adipokines for communication with other organs and tissues. The major process of lipogenesis is triacylglycerol synthesis which comprises such enzymes as monoacylglycerol acyltransferase and diglyceride acyltransferase for triacylglycerol storage in a form of lipid droplets. The other way around main enzymes of lipolysis adipose triglyceride lipase and hormone-sensitive lipase produce sufficient amount of energy for other tissues. Lipid combustion in brown adipose tissue produces heat in the body through the function of uncoupling protein 1. Signaling pathways of lipolysis and thermogenesis comprise adrenergic receptors. Study of thermogenic function of uncoupling protein and adipose tissue metabolism can be useful for the treatment of obesity and metabolic disorders.
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Molecular mechanism of carcinogenicity of aristolochic acid
Levová, Kateřina ; Stiborová, Marie (advisor) ; Ryšlavá, Helena (referee) ; Souček, Pavel (referee)
Aristolochic acids (AA) are carcinogenic and nephrotoxic alkaloids from Aristolochia species. Aristolochic acid I (AAI), the major component of AA, causes the development of Aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). These two diseases cause total renal failure and urothelial malignancies. The fact that these diseases have not been developed in all persons, who have been exposed to their action, might be causd by different activities and protein levels of the enzymes metabolizing AAI. Thus, the identification of enzymes involved in the metabolism, and detailed knowledge of their expression and catalytic specifities is a major importance. Aristolochic acid I (AAI) can be metabolized by several types of reactions. Like most nitroaromatics, the main activation pathway of AAI is reduction of its nitro group to form a cyclic acylnitrenium ion, which can bind to the purine bases, thereby forming AAI-DNA adducts. The detoxication pathway of AAI is its oxidative demethylation by cytochromes P450 forming detoxication metabolite 8-hydroxyaristolochic acid Ia (AAIa). In the present thesis, using rat and human enzymes and as well as several mice models, the metabolism of AAI in vitro and in vivo was investigated. The first model has deleted gene for NADPH:cytochrome P450...
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Adipose tissue metabolism and genetically modified murine models
Irodenko, Ilariia ; Bardová, Kristina (advisor) ; Železná, Blanka (referee)
Adipose tissue plays an important role in energy and glucose homeostasis. Adipose tissue metabolism includes lipolysis and lipogenesis processes which control lipid mobilization, storage and distribution in the body. In addition to that adipose tissue is recognized as an endocrine organ which generates cytokines and adipokines for communication with other organs and tissues. The major process of lipogenesis is triacylglycerol synthesis which comprises such enzymes as monoacylglycerol acyltransferase and diglyceride acyltransferase for triacylglycerol storage in a form of lipid droplets. The other way around main enzymes of lipolysis adipose triglyceride lipase and hormone-sensitive lipase produce sufficient amount of energy for other tissues. Lipid combustion in brown adipose tissue produces heat in the body through the function of uncoupling protein 1. Signaling pathways of lipolysis and thermogenesis comprise adrenergic receptors. Study of thermogenic function of uncoupling protein and adipose tissue metabolism can be useful for the treatment of obesity and metabolic disorders.
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SWI2/SNF2 ATPases with a focus on the ISWI subfamily: protein complexes and mouse models for their study
Turková, Tereza ; Stopka, Tomáš (advisor) ; Janoštiak, Radoslav (referee)
In the nucleus the DNA is packed along with proteins into a dynamic structure called chromatin. During cell cycle the chromatin structure becomes a subject to various changes. During interphase chromatin structure becomes loose while shortly before cell division it undertakes the form of highly condensed mitotic chromosomes. Structure of chromatin influences significantly mode of gene expression and its pattern. DNA-binding proteins interacting within chromatin are also necessary during this process. To gain the access to the DNA binding factors, the chromatin has to be in a loosened form. As long as the structure of the chromatin is more condensed it creates a barrier for the DNA binding proteins. Therefore it becomes obvious that the remodeling of the chromatin structure is one of the important regulators of gene expression and that the enzymes, which execute remodeling, are of great importance. One of them is ATPase Smarca5, which belongs to the protein subfamily ISWI and which creates the catalytic subunit for several different ATP-dependent chromatin remodeling complexes. Mutations of members of those complexes disturb regulation of transcription and cellular differentiation. In some cases the incorrect function of these complexes can lead to cellular transformation into a tumours state. This...
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Intestinal cancer and mouse models of the disease
Baloghová, Nikol ; Janečková, Lucie (advisor) ; Gemperle, Jakub (referee)
Intestinal cancer is a serious and common disease. To understand the mechanisms of its development, it is important to know the structure of the intestinal epithelium, as well as the signalling pathways that maintain the homeostasis and regulate cell proliferation and differentiation. Development of the intestinal cancer is a multistep process in which many molecular events underlie initiation and progression of the disease. Transgenic mice produced by genetic engineering are essential tools in both research of the intestinal cancer initiation and progression and possible treatment strategies. The aim of this work is to describe the intestinal anatomy and the renewal of the intestinal epithelium including the role of multiple signalling pathways, to summarize the most common mutations conditioning human colorectal carcinoma development and to define the existing mouse models of the disease.
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Molecular mechanism of carcinogenicity of aristolochic acid
Levová, Kateřina ; Stiborová, Marie (advisor) ; Ryšlavá, Helena (referee) ; Souček, Pavel (referee)
Aristolochic acids (AA) are carcinogenic and nephrotoxic alkaloids from Aristolochia species. Aristolochic acid I (AAI), the major component of AA, causes the development of Aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). These two diseases cause total renal failure and urothelial malignancies. The fact that these diseases have not been developed in all persons, who have been exposed to their action, might be causd by different activities and protein levels of the enzymes metabolizing AAI. Thus, the identification of enzymes involved in the metabolism, and detailed knowledge of their expression and catalytic specifities is a major importance. Aristolochic acid I (AAI) can be metabolized by several types of reactions. Like most nitroaromatics, the main activation pathway of AAI is reduction of its nitro group to form a cyclic acylnitrenium ion, which can bind to the purine bases, thereby forming AAI-DNA adducts. The detoxication pathway of AAI is its oxidative demethylation by cytochromes P450 forming detoxication metabolite 8-hydroxyaristolochic acid Ia (AAIa). In the present thesis, using rat and human enzymes and as well as several mice models, the metabolism of AAI in vitro and in vivo was investigated. The first model has deleted gene for NADPH:cytochrome P450...
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