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Detection of nestin in the developing muscle and injury induced in vivo using a model of skeletal muscle lesion
Pechancová, Adéla ; Němečková, Ivana (advisor) ; Čížková, Dana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Author: Adéla Pechancová Title of bachelor's thesis: Detection of nestin during development and injury with the use of in vivo model of skeletal muscle lesion Supervisor: RNDr. Ivana Němečková, PhD. Introduction and the aim of the study: Regeneration of tissues and organs is recently very actual topic. Regenerative medicine is coming to the foreground, it is a medical field focused on reparation of tissues. Intermediate filament nestin, which is considered to be a marker of stem cells and progenitor cells especially in nerve tissue, was also detected in immature elements of muscle tissue. The aim of this study was to prove expression of nestin during development and injury of skeletal muscle after injection of cardiotoxin and it's following regeneration using immunohistochemical methods. Methods: In the experimental part we were detecting nestin on paraffin embedded sections using mainly indirect immunohistochemical methods (LSAB, EnVision FLEX kit). The goal for the paraffin embedded section of mouse embryo was to capture the development of skeletal muscle. For the paraffin embedded section of leg muscle injured by cardiotoxin, the goal was to capture the amount of damage of the skeletal muscle and it's following ability to...
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Introduction of an invertebrate animal model, Galleria Galleria mellonellamellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics
Kubíček, Jiří ; Konečná, Klára (advisor) ; Němečková, Ivana (referee)
Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Jiří Kubíček Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Introduction of an invertebrate animal model, Galleria mellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics. Background: The aim of this diploma thesis has been to present an invertebrate organism, Galleria mellonella (G. mellonella), Pyralidae, as an alternative, more economically advantegous and morally more acceptable model for in vivo studies of an antituberculotics (anti-TB) efficacy. In the experimental part of this work, attention was paid to the optimization of methodological steps, that should lead to a higher reproducibility and validity of the obtained data. The effect of the disaggregating supplement, tyloxapol, on the growth of Mycobacterium aurum (M. aurum) was evaluated. The growth of this microbial strain in the culture medium, Middlebrook 7H9, was also mapped to determine the exponential growth phase. In the second part of the experiments, G. mellonella larvae were infected with a M. aurum bacterial strain, followed by an administration of the selected, commercially available anti-TB. Methods: To map the exponential phase of M....
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Carotuximab effects on inflammation during liver fibrosis development
Sharif, Navid ; Rathouská, Jana (advisor) ; Němečková, Ivana (referee)
Chronic liver inflammation is a process that results in the distortion of the liver parenchyma. Constant damage caused by inflammation would lead to formation of fibrotic tissue which can impair the normal liver physiology. Liver fibrosis is one of the common causes of morbidity and mortality worldwide. Non-alcoholic fatty liver disease (NAFLD) is a pathological state that is characterised by excessive accumulation of lipid in the hepatocytes (hepatic steatosis) which can trigger the inflammatory response in the liver. Carotuximab (TRC 105) is a monoclonal antibody that has been used for the treatment of a number of cancers, including hepatocellular carcinoma (HCC). The aim of this study was to investigate the role of carotuximab in liver inflammation in a mouse model of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis. Eighteen 3-month-old C57BL/6 male mice were recruited in this study. They were divided into three groups: control group (n=6) - fed chow diet for the period of 4 weeks, DDC group (n=6) - fed DDC diet for the period of 4 weeks, DDC group treated with carotuximab (DDC+TRC, n=6) - fed DDC diet for the period of 4 weeks and to that TRC 105 (15mg/kg) was administered. The impact of the diet and TRC 105 on mouse weight, as well as the effect on liver damage (levels...
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Immunohistochemical analysis of TRC105 effects on the liver fibrosis
Hamerníková, Veronika ; Němečková, Ivana (advisor) ; Svobodová, Zuzana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biological and Medicinal Sciences Studijní obor: Pharmacy Author: Veronika Hamerníková Supervisor: RNDr. Ivana Němečková, Ph.D. Title of master thesis: Immunohistochemical analysis of TRC105 effects on the liver fibrosis The subject of this diploma thesis was the analysis of the expression of liver fibrosis biomarkers in mice liver. The aim of our work was to test the effect of the drug TRC105 (carotuximab) on a fibrotic liver model after 4 weeks of DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-colidine) administration. Collagen deposition and expression of α-SMA, GFAP and endoglin were examined and compared. Animals were divided into 3 groups: control group (n=6), DDC group (n=6) and TRC105 group (n=6). The control group was fed a standard diet, while the DDC and the TRC105 groups were fed a diet with 0.1% DDC. Physiological solution was administered to the control and DDC groups while carotuximab was administered to the TRC105 group. The analysis of fibrosis biomarker expression was then performed on fixed liver sections. Collagen expression was evaluated by Picro-Sirius Red staining and α-SMA, GFAP and endoglin expression by immunohistochemistry with fluorescence imaging. Histological staining of collagen...
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Histological image of healthy liver and comparison with liver steatosis and cirrhosis.
Čulíková, KIára ; Svobodová, Zuzana (advisor) ; Němečková, Ivana (referee)
The liver is a very important parenchymatous organ for humans. Their function can be impaired by various influences. Common noxins include hepatotropic viruses and other microorganisms, alcohol and other toxins, including drugs, metabolic disorders, biliary obstruction, tumors, genetic abnormalities, and others. The work focuses on the comparison of histological images and clinical diagnosis of healthy individuals with steatosis and cirrhosis. The theoretical part describes the anatomy, physiology and histology of the liver with the perihepatic area. The next chapters focus on the clinic of steatosis and cirrhosis. The practical part describes the procedures for the preparation of healthy and affected liver preparations, as well as the histological methods used for staining individual cell structures. The result of comparing diagnoses of histological specimens and clinical diagnoses of individuals is 94.23 % agreement. Although the found discrepancy of diagnoses is small (5.77 %), it is a reason to think about the development and use of new or modified methods, including the creation of various calculators, including clinical data. Key words: histology, liver, steatosis, cirrhosis
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Histopathological alterations in lungs associated with COVID-19
Šindelářová, Valerie ; Němečková, Ivana (advisor) ; Nachtigal, Petr (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biological and Medical Sciences Study program: Laboratory diagnostics in healthcare Author: Valerie Šindelářová, Dis. Supervisior: RNDr. Ivana Němečková, Ph.D. Title of Thesis: Histopathological alterations in lungs associated with COVID-19 The bachelor thesis is focused on histopathological alterations in the lungs associated with viral disease COVID-19. The theoretical part deals with anatomy, histology and lung function. There is also an overview of lung diseases caused by viral and bacterial agents. The main part is devoted to COVID-19 disease and histopathological methods of necropsy material processing. The practical part presents individual case reports - patients in whom COVID-19 was identified as the main cause of death and in whom this disease left visible changes in lung tissue. A preparation of the physiological lungs and the lungs of a patient with bronchopneumonia is also included here for comparison. COVID-19 is an infectious respiratory disease caused by the SARS-CoV-2 virus, which affects the whole organism. However, it most often causes serious respiratory diseases - such as pneumonia, lung malfunction or acute respiratory distress syndrome. This bachelor's thesis is focused exactly on...
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Complex morphological, immunohistochemical, molecular genetic and clinical pathological analysis of rare types of ovarian cancers
Galko, Kamila ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Ovarian carcinomas are heterogenous group of diseases comprising various histotypes. Ovarian carcinomas as a whole are associated with the highest lethality among the female genital tumors. Currently, 5 main types of carcinomas are recognized: high grade serous carcinoma, low grade serous carcinoma, endometroid carcinoma, clear cell carcinoma and mucinous carcinoma. Other types, e.g. Brenner tumors, are very rare. Ovarian carcinomas are very diverse group with various precursors, morphology, genetic aberrations, epidemiologic and clinical, features, therapy and prognosis. The most studied and understood is high grade serous carcinoma with high incidence (up to 70 % of ovarian carcinomas). The number of studies dedicated to this tumor is still increasing. Other types of carcinomas are much less frequent, therefore related studies are rarer. Due to the rapid development of targeted therapy, it is necessary to evaluate the tumors not only morphologically but also on immunohistochemical and genetic level. Detection of neoantigens may indicate the mutation load of a tumor, it is also possible to evaluate status of mismatch repair (MMR) proteins and other features leading to chromosomal aberrations. In contrast to the high grade serous carcinoma, as for other types of carcinomas, knowledge about their...
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Paraffin immunofluorescence, an ancillary technique in renal pathology - one center study
Buchová, Karolína ; Němečková, Ivana (advisor) ; Kudláčková, Zděnka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biological and Medical Sciences Study program: Bioanalytical laboratory diagnostics in healthcare Author: Bc. Karolína Buchová Supervisior: RNDr. Ivana Němečková, Ph.D. Consultant: doc. MUDr. Kristýna Pivovarčíková, Ph.D. Title of Thesis: Paraffin immunofluorescence, an ancillary technique in renal pathology - one center study Background: Immunofluorescence analysis (IFLU) is routinely used to diagnose non-tumor renal diseases, and also has a place in renal transplant pathology. This examination is most often performed on frozen sections from non-fixed tissue. The next option where we could use the IFLU method is examination in formaldehyde-fixed tissues which are embedded in paraffin blocks. However, using the formaldehyde-fixed paraffin-embeded samples has its specifics and require a modification of the methodology and verification. This diploma thesis follows the differences in the processing of renal punctures when we use IFLU for frozen material (FIF) or IFLU for formalin-fixed and paraffin- embedded tissue (PIF) and comparison of the results obtained by both methods. Main findings: Immunofluorescence examination is very important part in examination of kidney biopsy obtained by puncture. The...
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Carotuximab effects on inflammation during liver fibrosis development
Sharif, Navid ; Rathouská, Jana (advisor) ; Němečková, Ivana (referee)
Chronic liver inflammation is a process that results in the distortion of the liver parenchyma. Constant damage caused by inflammation would lead to formation of fibrotic tissue which can impair the normal liver physiology. Liver fibrosis is one of the common causes of morbidity and mortality worldwide. Non-alcoholic fatty liver disease (NAFLD) is a pathological state that is characterised by excessive accumulation of lipid in the hepatocytes (hepatic steatosis) which can trigger the inflammatory response in the liver. Carotuximab (TRC 105) is a monoclonal antibody that has been used for the treatment of a number of cancers, including hepatocellular carcinoma (HCC). The aim of this study was to investigate the role of carotuximab in liver inflammation in a mouse model of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis. Eighteen 3-month-old C57BL/6 male mice were recruited in this study. They were divided into three groups: control group (n=6) - fed chow diet for the period of 4 weeks, DDC group (n=6) - fed DDC diet for the period of 4 weeks, DDC group treated with carotuximab (DDC+TRC, n=6) - fed DDC diet for the period of 4 weeks and to that TRC 105 (15mg/kg) was administered. The impact of the diet and TRC 105 on mouse weight, as well as the effect on liver damage (levels...
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Impact of various fixation methods on bone marrow trepanobiopsies processing
Panócová, Miroslava ; Němečková, Ivana (advisor) ; Fikrová, Petra (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of Diploma Thesis: Impact of various fixation methods on bone marrow trepanobiopsies processing Author: Bc. Miroslava Panócová Supervisor of Diploma Thesis: RNDr. Ivana Němečková, PhD. Background: The aim of this diploma thesis is to determinate whether the length of fixation influences the processing of bone marrow trepanobiopsies. Methods: This study was performed on twelve bone marrow trepanobiopsy specimens fixed in 10% neutral buffered formalin (NBF). The samples were divided into two groups based on diagnosis. Three fixation lengths (24 hours, 48 hours, and more than 48 hours) were examined. The fixed samples were processed in the laboratory as standard, stained in a Shandon Varistain Gemini staining machine and handed over to a clinical pathologist for evaluation. Results: Individual fixation lengths achieved the following laboratory technician/clinical pathologist scores: the 24-hour fixation score was 6.71/7.71, the 48-hour fixation score was 6.57/7.71, and the more than 48-hour fixation score was 7.42/8.42. In terms of division into groups, in group C, the 24-hour fixation is rated at 6/7.66, the 48-hour fixation at 6.33/7.66, and the more than 48-hour fixation at 8.33/9. In group...
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