Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Tereza Kněžíková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Liver fluke - treatment and resistence Fasciola hepatica is a parasite of global importance that we find both in farm animals and in humans. This thesis aims to summarize information on the potential of drugs and treatment alternatives that are suitable for treatment of F. hepatica. Given that for a number of drugs used in the past, as well as the drugs currently administered, resistance developed, this thesis is also focused on this phenomenon, especially the mechanisms of its origin. The drugs used to treat fasciolosis are called antitrematodal drugs. They can be divided into five chemical groups, of which the most important group are currently benzimidazoles and their representative triclabendazole. Also other drugs as albendazole, clorsulon, hexachlorophene, closantel, diamphenitide, bithionol, rafoxanide are important. The rate of resistance development is affected by many factors that may be genetic, biological or functional. F. hepatica actively uses its enzymatic system, especially oxidation enzymes or efflux transporters. The influence on the development of resistance, apart from the parasite itself,...

Myofibroblast expansion is a critical event in the pathogenesis of liver fibrosis. The activation of hepatic stellate cells (HSC) to myofibroblast (MFB) results in the enhanced production of extracellular matrix (ECM). We have studied the effect of fibroblast growth factor 1 (FGF-1) on liver MFB. In the second part we investigated effect of transforming growth factor β1 (TGF-β1) and FGF-1 on cell line HSC-T6. Cells were cultured on plastic dishes and in 3D collagen gel mimicking fibrotic tissue. MFB were isolated by repeated passaging of nonparenchymal liver cell fraction. The transfer of MFB from plastic dishes to collagen gel resulted in the change in their shape and phenotype. The expression of cytokine TGF-β1 and of MFB markers, α-smooth muscle actin (α-SMA) and cellular fibronectin (EDA-FN) on protein level was significantly decreased in collagen gel. The experiments with SB 431542, the inhibitor of TGF-β receptor type I, showed that EDA-FN and α-SMA are differently regulated. EDA-FN expression is dependent on TGF-β1, while the expression of α-SMA is primarily determined by the environment and modified by TGF-β1. EDA-FN is more sensitive to the U0126, the inhibitor of protein kinases MEK 1 and 2. Collagen gel does not change the expression of metalloproteinase MMP-2 but activates the proenzyme....

Introduction: The reason for the inability of performing the liver resection for colorectal carcinoma metastasis is usually insufficient remnant liver parenchyma after liver resection (future liver remnant volume - FLRV). The current standard method of increasing FLRV is the embolization of the branch of portal vein (portal vein embolization - PVE) on the side of the tumor, and then suspended after hypertrophy of the non-embolised lobe liver resection. Unfortunately, there are some patients who do not increase liver volume despite perfectly executed PVE. Besides that, FLRV occurs during the time necessary for hypertrophy progression of metastatic disease. Therefore, we are trying to find the appropriate way to encourage the growth of remaining liver parenchyma and accelerate hypertrophy of the contralateral liver lobe. From our previous experience (IGA MZ NS 10240), it is possible to be optimistic that there hope is the way of hematopoietic progenitor cells (HPC - adult stem cells) after previous PVE to non-embolised branches of the portal vein. These cells do not only accelerate liver regeneration, but are also able to improve its function (function of the liver) which is especially important for patients after neoadjuvant chemotherapy (steatohepatitis or steatofibrosis), and for patients with...

My thesis consists of a theoretical part and a practical part. In theoretical part I deal with information about the fatty acids and its structure, functions and influence on our health. Last but not least I focused on diseases that may arise due to bad intake of fatty acids. It is the atherosclerosis. The second part of my thesis is the already mentioned practical part. This consisted of a samples of pigs in which were taken all animal tissues and subsequent determination of the fatty acids. In my thesis I deal with liver and muscle tissue. These samples were taken from pigs that had fish or palm oil in the diet. The determination was done by lyophilization, extraction, derivatization and gas chromatography. Based on the results, I conducted a statistical evaluation using graphs and a final evaluation of the results.

In this thesis, the impact of atenolol, verapamil, dexamethasone, clotrimazole, and PBSA on the physiological condition and health of rainbow trout (Oncorhynchus mykiss) were studied in vivo and in vitro. The sub-chronic test of atenolol, including at the environmentally relevant concentration, demonstrated that only the fish exposed to atenolol at a concentration of 1000 ?g?L-1 may be at a higher risk of oxidative stress. The second pharmaceutical compound studied was an L-type calcium channel blocker, verapamil. Despite the relatively high concentration of verapamil included in the experiments, the rates of seven hepatic CYP450-mediated reactions (EROD, MROD, PROD, BFCOD, COH, and PNPH) were unchanged in the exposed rainbow trout. This indicates that the activities of the selected CYP450 enzymes were not affected. Exposure to the highest concentration of PBSA (1000 ?g?L-1) led to significantly inhibited glutathione reductase activity. The activities of the EROD, MROD, and PROD enzymes gradually increased, beginning at the environmentally relevant concentrations. We suggest that long-term exposure to PBSA could cause oxidative stress in fish, as indicated by the decreased activity of GR. Moreover, PBSA could interact with intracellular receptors due to the elevated activities of CYP1A (EROD and MROD) and CYP2B (PROD). Rainbow trout showed multiple responses after exposure to clotrimazole. Changes in the haematological parameters in the exposed rainbow trout suggested that the immune system and haemoglobin synthesis were affected. The antioxidant defence enzymes that responded to the clotrimazole exposure influenced the oxidative stress status of the fish. Sub-chronic exposure to clotrimazole did not lead to oxidative stress in any of the fish tissues studied, as indicated by the unchanged TBARS levels. Hepatic energetic pathways were activated in the tested groups after 42 days of exposure to clotrimazole. High levels of glutathione S-transferase were seen in the liver and gill tissues, possibly associated with the detoxification of clotrimazole. The interaction of clotrimazole with intracellular receptors can be expected due to the changes in the catalytic activities of CYP1A1 and CYP3A. The BCF levels in the muscle tissue suggest a moderate bioaccumulation of CLO. The relatively long elimination period for CLO in the fish kidney (half-life = 29 days) may imply a greater potential for adverse effects in this organ. This finding is significant in view of the structural disturbances that appeared in the kidney and testis. The changes observed in the kidney were most prominent in the tubular epithelial cells. Changes in the gonads were only visible in the male specimens, and the pathological changes in the testis may indicate a disruption of steroidogenesis. The potential of clotrimazole and dexamethasone to inhibit CYP450 enzyme activity in rainbow trout hepatic microsomes was investigated in vitro. Clotrimazole non-competitively inhibited EROD activity in the hepatic microsomes, while BFCOD activity was competitively inhibited. PNPH activity was only slightly reduced in the presence of clotrimazole, indicating that it does not significantly interact with CYP2E1. The presence of dexamethasone in the incubations did not affect any of the CYP450 enzymes investigated. Everything considered, the results of the study confirmed that the presence of clotrimazole in the environment is of concern with respect to its impact on the health status of fish. In this thesis, the data of the actual effects of several PPCPs on fish were discussed. Based on the response of selected molecular endpoints, the cardiovascular and anti-inflammatory drugs studied do not present a significant risk for fish, while the antifungal drug and UV-screening agent that were tested may disrupt several physiological processes in fish.

Dihydromyricetin (DMH) is a natural flavonoid compound with positive effects on the human organism. In traditional Chinese medicine, plants containing DMH were used to treat liver diseases and to reduce alcohol intoxication. The effects of DHM on ethanol metabolism are not yet completely understood. Effects of DHM during alcohol intoxication were studied on primary hepatocytes of rats. DCFDA and DHR probes were used to prove that DHM (depending on concentration) reduces the number of reactive oxygen and nitrogen species in primary hepatocytes. However, the hepatoprotective effects of DHM were not achieved when presence of the alanine aminotransferase (ALT) was used to measure the damage of cells exposed to alcohol. Further, the effects of DHM on alcohol metabolism were studied in vivo. Rats were administered with single dose of ethanol or ethanol combined with DHM. Measured blood levels of ethanol and acetaldehyde show that DHM has no effects on the rate or levels of alcohol metabolism. The effects of DHM were also studied with repeated alcohol administration. In the group that was administered also DHM, increased blood levels of ethanol were measured. This points that DHM slow down the metabolic rate of ethanol. Obtained results did not prove any positive effects of DHM on alcohol metabolism....

This thesis is aimed at studying the effect of the addition of milk thistle (Silybum marianum) seed cakes to the diet on metabolism, liver function and performance of laying hens at the beginning of laying. In the experiment, the results were compared between a group of laying hens, which were fed with the basic feed mixture without the addition of milk thistle seed cakes with the experimental group with the addition of milk thistle seed cakes in the feed mixture. It was situated 30 hens breed ISA Brown into the experiment, divided into two groups. The first group was experimental with the addition of milk thistle seed cakes (7 % in the mixture), and the second group was a control. The experiment evaluated egg laying, egg weight, food consumption and the animal weight. During the experiment, blood was twice collected from animals for biochemical examination focusing on the parameters of liver profile. The results of the experiments didn´t show any effects of feeding milk thistle seed cakes on the body weight of laying hens. By evaluating the performance parameters of the laying hens it showed a significantly lower egg laying, but also showed a lower consumption of feed (P < 0.05). At the same time, the data found higher weigh of eggs (P < 0.01) in this group, but the egg production was significantly lower than in the control group. During the examination of biochemical parameters of blood there was a significantly higher concentration of bilirubin, uric acid and cholesterol (P < 0.05) in the blood plasma of laying hens fed by the addition of milk thistle seed cakes. At the second sampling (approximately 2 months) the laying hens that were fed with milk thistle seed cakes have consistently higher concentration of cholesterol (P < 0.05) and lower activity of dehydrogenase, creatine kinase (P < 0.05) and uric acid (P < 0.01) in the blood. In the experiment there was no clear positive or negative effects of the milk thistle seed cakes on the performance parameters and biochemical profile of the blood plasma.

Introduction: The reason for the inability of performing the liver resection for colorectal carcinoma metastasis is usually insufficient remnant liver parenchyma after liver resection (future liver remnant volume - FLRV). The current standard method of increasing FLRV is the embolization of the branch of portal vein (portal vein embolization - PVE) on the side of the tumor, and then suspended after hypertrophy of the non-embolised lobe liver resection. Unfortunately, there are some patients who do not increase liver volume despite perfectly executed PVE. Besides that, FLRV occurs during the time necessary for hypertrophy progression of metastatic disease. Therefore, we are trying to find the appropriate way to encourage the growth of remaining liver parenchyma and accelerate hypertrophy of the contralateral liver lobe. From our previous experience (IGA MZ NS 10240), it is possible to be optimistic that there hope is the way of hematopoietic progenitor cells (HPC - adult stem cells) after previous PVE to non-embolised branches of the portal vein. These cells do not only accelerate liver regeneration, but are also able to improve its function (function of the liver) which is especially important for patients after neoadjuvant chemotherapy (steatohepatitis or steatofibrosis), and for patients with...

The liver fluke, Fasciola hepatica, is one of the most important parasites of livestock, and it also infects humans. The proteolytic system of trematodes is critical for their interaction with the host and is a potential target for the development of novel vaccines. This work is focused on proteases secreted by F. hepatica adults and on FheCy2, a new protease inhibitor from the cystatin family. The proteolytic activity of the secreted proteases was analyzed using: (a) chromogenic protein substrates and fluorogenic peptide substrates, (b) selective protease inhibitors, and (c) a fluorescent activity-based probe for visualization of proteases. The results showed that the secreted proteases are cysteine proteases of papain family belonging to cathepsins L and B. These proteases were effectivelly inhibited by FheCy2 as demonstrated by enzymological analysis. It can be assumed that FheCy2 participates in the physiological regulation of endogenous proteases secreted by F. hepatica adults, which makes it attractive candidate protein for vaccination studies. Key words: Fasciola hepatica, cathepsins, proteolytic activity, substrate specificity, protease inhibitors (In Czech)

We tested the hypothesis that triacylglycerol (TAG) accumulation in the liver induced by short-term high-fat diet (HFD) in rats leads to the dysregulation of endogenous TAG degradation via lysosomal pathway and is causally linked with the development of hepatic insulin resistance. Lysosomal lipase (LAL) is stored in qualitatively different depots (light and dense lysosomes). In contrast to dense lysosomal fraction, LAL associated with light lysosomes exhibits high activity on intracellular TAG and prandial- or diet-dependent regulation. On standard diet, LAL activity was up-regulated in starved and down-regulated in fed animals. In the HFD group, we demonstrated elevated LAL activity, increased TAG content, enhanced production of diacylglycerol and the abolishment of prandial-dependent LAL regulation in light lysosomal fraction. The impairment of insulin signalling and increased activation of PKCε was found in liver of HFD-fed animals. Lipolysis of intracellular TAG, mediated by LAL, is increased in steatosis probably due to the enhanced formation of phagolysosomes. Consequent overproduction of diacylglycerol may represent the causal link between HFD-induced hepatic TAG accumulation and hepatic insulin resistance via PKCε activation.