The Study of Mechanisms of Hepatotoxic and Hepatoprotective Effects in a Model of Damaged Hepatocytes

Liver damage has a variety of causes. Due to the role of liver in the metabolism of xenobiotics, liver damage is often caused by toxic substances. Despite the variety of causes, the mechanisms of damage may be common. Most often, oxidative stress is induced in the cell or important organelles are directly damaged. This leads to cell death, the initiation of an inflammatory response and the development of fibrosis. A major problem in Western civilization is Non Alcoholic Fatty Liver Disease (NAFLD), in which fat is stored in liver tissue. Steatotic hepatocytes may be more sensitive to damage compared to non-fatty ones. The study of hepatotoxicity and possible hepatoprotection requires suitable models for in vitro experiments. The first part of this work is therefore dedicated to the introduction of a model background using hepatocytes isolated from rat liver. First, we introduced a method of culturing hepatocytes in a sandwich and evaluated its benefit over cultivation in a monolayer, even for experiments lasting 48 hours, which are frequently used in our laboratory. Significant differences were recorded from day 4 of culture, when the sandwich model provides better preservation of morphological and functional parameters of hepatocytes. However, for our purposes of studying the mechanisms of..

Evaluation of peritoneal macrophage activity

Tato diplomová práce se zabývá stanovením tří cytokinů produkovaných peritoneálními makrofágy potkana - TNF?, IL-1? a IL-6 ve vztahu k rozdílnému přístupu operačního výkonu - laparotomickému či laparoskopickému. Byla měřena bazální produkce uvedených cytokinů a jejich produkce po stimulaci makrofágů bakteriálním lipopolysacharidem (LPS). Izolace buněk z peritonea probíhala ve dvou intervalech - 24 nebo 72 hodin po operaci. Vlastní analýze předcházela vždy 24 hodinová kultivace s, resp. bez LPS. Optimální koncentrace LPS vhodné pro stimulaci peritoneálních makrofágů bylo nutno experimentálně docílit. Zjištěná optimální koncentrace LPS byla 0,1 ?g/ml. Hladina cytokinů byla stanovena pomocí sendvičové ELISA metody. V teoretické části je popsána organizace imunitního systému a funkce jeho jednotlivých složek v imunitní odpovědi s důrazem na úlohu makrofágů. Významná část je věnována roli cytokinů v modulaci imunitní odpovědi. Dále je podána literární rešerše o dějích v peritoneální oblasti ve vztahu k laparotomickému či laparoskopickému zásahu do abdominální krajiny a zhodnocení systémové a lokální imunitní odpovědi, která tyto chirurgické zákroky doprovází. Experimentální část je věnována popisu principu EIA metod se zaměřením na ELISA metody a její variace. Dále je v této části uveden optimalizovaný postup izolace a kultivace peritoneálních makrofágů a vlastního stanovení jimi produkovaných cytokinů metodou ELISA. Výsledková část obsahuje postup optimalizace koncentrace LPS vhodného pro stimulaci peritoneálních makrofágů. Dále zahrnuje grafickou a slovní dokumentaci bazální i stimulované produkce jednotlivých cytokinů makrofágy izolovanými 24, resp. 72 hodin po zákroku. Bazální produkce cytokinů byla signifikantně vyšší u laparoskopované skupiny, nejvyšší produkci cytokinů stimulovanými makrofágy vykazovala naopak skupina po laparotomii. Zhodnocení získaných výsledků je rozebíráno v diskusi.This thesis deals with the determination of the three cytokines produced by rat peritoneal macrophages - TNF?, IL-1? and IL-6 in relation to the different approach of surgery - laparoscopic or laparotomic. Was measured basal production of cytokines and those of their production after stimulation of macrophages by bacterial lipopolysaccharide (LPS). Isolation of peritoneal cells was performed in two intervals - 24 or 72 hours after surgery. The actual analysis is always preceded by 24 hours of cultivation with, respectively without LPS. The optimal concentration of LPS suitable for stimulation of peritoneal macrophages was necessary to achieve experimentally. The observed optimal concentration of LPS was 0,1 ?g/ml. The level of cytokines was determined using a sandwich ELISA method. The theoretical part describes the organization of the immune system and functions of its individual components in the immune response with emphasis on the role of macrophages. A significant part is devoted to the role of cytokines in the modulation of immune responses. In addition, is given a literature review about actions in the peritoneal area in relation to laparotomic or laparoscopic intervention into the abdominal landscape and assess systemic and local immune response that accompanies the surgery. The experimental section is devoted to describing the principle of EIA methods, focusing on the ELISA method and its variations. Additionally, the optimized process isolation and cultivation of peritoneal macrophages and optimized determination of cytokines measured by ELISA is described there. Results contain the process of optimization of an appropriate concentration of LPS suitable for stimulation of peritoneal macrophages. It also includes a graphical and verbal documentation of basal and stimulated production of cytokines by macrophages isolated 24, resp. 72 hours after surgery. Basal cytokine production was significantly higher in laparoscopic group, the highest cytokine production by stimulated macrophages showed a group after laparotomy. Evaluation of obtained results is discussed in the discussion.Katedra biologických a biochemických věd1) Prezentace výsledků diplomové práce 2) Diskuze k posudkům vedoucího a oponenta diplomové práce 3) Diplomantka zodpověděla všechny dotazy a připomínky k DPDokončená práce s úspěšnou obhajobo

The Study of Mechanisms of Hepatotoxic and Hepatoprotective Effects in a Model of Damaged Hepatocytes

Poškození jater má rozmanitou škálu příčin. V důsledku jejich postavení v metabolismu xenobiotik dochází často k poškození jater toxickými látkami s následnou poruchou funkcí. I přes různorodost příčin, mechanismy poškození mohou být společné. Nejčastěji dochází k indukci oxidačního stresu v buňce či k přímému poškození důležitých organel. To vede ke smrti buněk, iniciaci zánětlivé reakce a rozvoji fibrózy. Velikým problémem západní civilizace je nealkoholová tuková choroba jater (NAFLD - Non Alcoholic Fatty Liver Disease), při které dochází k ukládání tuků v jaterní tkáni. Steatotické hepatocyty mohou vykazovat vyšší senzitivitu k poškození v porovnání s neztukovatělými. Studium hepatotoxicity a možné hepatoprotekce vyžaduje pro in vitro experimenty vhodné modely. První částí této práce je proto příprava modelového zázemí s použitím hepatocytů izolovaných z jater potkana. Nejprve jsme zavedli metodu kultivace hepatocytů v sendviči a zhodnotili její přínos oproti kultivaci v monolayeru, a to i pro experimenty trvající 48 hodin, které jsou v naší laboratoři frekventovaně využívány. Výrazné rozdíly stavu hepatocytů byly zaznamenány od 4. dne kultivace, kdy sendvičový model poskytuje lepší zachování morfologických i funkčních parametrů hepatocytů. Nicméně, pro naše účely studia mechanismů...Liver damage has a variety of causes. Due to the role of liver in the metabolism of xenobiotics, liver damage is often caused by toxic substances. Despite the variety of causes, the mechanisms of damage may be common. Most often, oxidative stress is induced in the cell or important organelles are directly damaged. This leads to cell death, the initiation of an inflammatory response and the development of fibrosis. A major problem in Western civilization is Non Alcoholic Fatty Liver Disease (NAFLD), in which fat is stored in liver tissue. Steatotic hepatocytes may be more sensitive to damage compared to non-fatty ones. The study of hepatotoxicity and possible hepatoprotection requires suitable models for in vitro experiments. The first part of this work is therefore dedicated to the introduction of a model background using hepatocytes isolated from rat liver. First, we introduced a method of culturing hepatocytes in a sandwich and evaluated its benefit over cultivation in a monolayer, even for experiments lasting 48 hours, which are frequently used in our laboratory. Significant differences were recorded from day 4 of culture, when the sandwich model provides better preservation of morphological and functional parameters of hepatocytes. However, for our purposes of studying the mechanisms of...Department of PhysiologyÚstav fyziologieFaculty of Medicine in Hradec KrálovéLékařská fakulta v Hradci Králov

Financial Management of the Museum

Import 19/10/2011Tato práce je zaměřena na hospodaření příspěvkové organizace v oblasti kultury. První část se věnuje neziskovým organizacím. Mapuje postavení kultury ve státu. Zabývá se vymezením neziskových organizací a rovněž je definuje. Druhá část charakterizuje vybranou neziskovou organizaci, charakterizuje její principy hospodaření. Třetí část je věnována analýze hospodaření muzea jak v činnosti hlavní, tak i doplňkové. Hodnotí podíl doplňkové činnosti na celkovém podílu hospodaření organizace.This thesis is focused on the management of the contributory organisation in the culture sphere. The first part devotes to the non-profit organisations. It shows the position of culture in the country. It deals with the definition of non-profit organisations. The second part characterizes the chosen non-profit organisation, describes its management principles. The third part is based on the analysis of the museum management according to the main activity and also the supplementary. The thesis evaluates the share of the supplementary activity with the total contribution of the organisation management.153 - Katedra veřejné ekonomikyvýborn

Geography of the Local Region (using Ricany as an example)

The theme of this thesis is Local Region in Education at Primary Schools in Ricany. The thesis is divided into two main parts. The first part deals with changes in education and their influence on teaching Geography: how the methods, forms of education and topics have changed. There is also a description of how the local region is included in education. There is a description of the local region, and possibilities of using the "local region" topic in education. At the end of the first part there is the summary of general objectives of teaching local region topics. The second part offers worksheets. There are four worksheets for children with the topic Local Region Ricany. There are four different problems in the local region. In the first worksheet the question is: How to demark the local region? And in which administrative unit of the Czech Republic the local region is included? The second topic is the traffic location of the local region. The third one is about suburbanization and its problems and the last topic is the creation of tourist guide for different groups of tourists. Every worksheet includes characterization of the problem in Ricany region, guide to worksheet for teachers, specific targets of the exercises which the pupils should know after they have finished the worksheets and finally the..

Health and social care of clients with schizophrenia in the Vysočina region

Schizophrenia is a serious psychotic illness with a 1-1,5% prevalence. This illness is present in all cultures at all continents. The first goal of the practical part of this thesis is to find out what impact schizophrenia has on clients´ family, their social and working life as well as health and economical situation. The second goal is to find out how do clients with schizophrenia actually deal with problems brought by the illness

Pracovní spokojenost všeobecných sester na oddělení standardní a intenzivní péče: „Ta sestra je pro všechno!“

Nové trendy v oblasti poskytování zdravotní péče činí jeho prostředí dynamickým s rostoucími požadavky na zdravotnický personál. Za zásadní měřítko kvality poskytované péče je považována dostatečná kapacita a odborná vzdělanost zdravotnického personálu. Právě nedostatek zdravotnického personálu se stává v současné době jedním z palčivých témat a výzvou, jíž zdravotnický systém a celá společnost čelí. Cílem textu je objasnit, jak všeobecné sestry vnímají svoji profesi a co ovlivňuje míru jejich pracovní spokojenosti. Byl proveden kvalitativní výzkum. Metodou hloubkových rozhovorů byly zjišťovány zkušenosti všeobecných sester s výkonem povolání, které působily na oddělení standardní a intenzivní péče. Všeobecné sestry hodnotí problematičnost jejich práce skrze aspekty vztahující se zejména k managementu, týmové spolupráci, pracovním podmínkám, kompetencím a adaptačnímu procesu či k prestiži a úctě. Dochází ke vzájemnému prolínání se jednotlivých faktorů, které působí na subjektivitu všeobecných sester v různých situacích a v odlišné intenzitě. Všeobecné sestry vnímají vyšší finanční ohodnocení jako motivaci, nikoliv jako zásadní prediktor jejich pracovní spokojenosti. „Klíč“ ke spokojenému výkonu povolání byl sestrami interpretován převážně skrze komunikaci a spolupráci napříč všemi profesemi.New trends in providing healthcare are dynamically changing the field and increasing demands on healthcare staff. The adequate capacity and training of healthcare staff is considered an essential measure of the quality of the provided care. The shortage of health personnel is becoming one of the most pressing issues and challenges facing both the health system and society as a whole. The aim of this text is to shed light on how general nurses perceive their profession and what influences their level of job satisfaction. To this end, qualitative research was conducted and the method of in-depth interviews was used to determine the experiences of general nurses (working in standard and intensive care units) in their profession. The general nurses evaluated the issues of their work through aspects related mainly to management, teamwork, working conditions, competency, adaptation process, prestige and respect. There is an interplay between the various factors that affect the subjectivity of general nurses in different situations and with different intensities. General nurses perceive higher financial remuneration as a motivation, but not as a major predictor of their job satisfaction. Nurses interpreted the key factors for job satisfaction to be communication and collaboration

ANTIOXIDATIVE EFFECT OF EPIGALLOCATECHIN GALLATE AGAINST D-GALACTOSAMINE-INDUCED INJURY IN PRIMARY CULTURE OF RAT HEPATOCYTES

Literature data support that green tea and its major component epigallocatechin gallate (EGCG) have powerful antioxidant effects. Contrary, hepatotoxicity can be induced by high-dose EGCG. The timing of exposure to green tea in relation to administration of hepatotoxic agent plays an import role too. The aim of our work was a verification of antioxidative effect of EGCG on D-galactosamine-induced injury in primary culture of rat hepatocytes. Hepatocytes were incubated with EGCG at concentrations of 1.25–10 μM and toxic D-galactosamine (GalN) for 24 hrs. Alternatively, hepatocytes were pretreated with EGCG for 24 hrs, and then incubated with EGCG and GalN for further 24 hrs. Cytotoxicity was analysed by lactate dehydrogenase activity, functional capacity by albumin production. Oxidative stress was evaluated from a production of malondialdehyde and glutathione content in the cells. EGCG protected hepatocytes against GalN-induced cytotoxicity but preventive treatment of intact hepatocytes with EGCG was required to diminish the development of hepatocyte injury. Oxidative stress induced in our study seems to overcome the ability of hepatocytes to improve GSH depletion and albumin production. Prolongation of the pretreatment with EGCG could be a promising strategy leading to amelioration of its hepatoprotective effect

Sulfated Metabolites of Flavonolignans and 2,3-Dehydroflavonolignans: Preparation and Properties

Silymarin, an extract from milk thistle (Silybum marianum) fruits, is consumed in various food supplements. The metabolism of silymarin flavonolignans in mammals is complex, the exact structure of their metabolites still remains partly unclear and standards are not commercially available. This work is focused on the preparation of sulfated metabolites of silymarin flavonolignans. Sulfated flavonolignans were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate as a sulfate donor and characterized by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). Their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging; ferric (FRAP) and Folin–Ciocalteu reagent (FCR) reducing activity; anti-lipoperoxidant potential; and effect on the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway were examined. Pure silybin A 20-O-sulfate, silybin B 20-O-sulfate, 2,3-dehydrosilybin-20-O-sulfate, 2,3-dehydrosilybin-7,20-di-O-sulfate, silychristin-19-O-sulfate, 2,3-dehydrosilychristin-19-O-sulfate, and silydianin-19-O-sulfate were prepared and fully characterized. Sulfated 2,3-dehydroderivatives were more active in FCR and FRAP assays than the parent compounds, and remaining sulfates were less active chemoprotectants. The sulfated flavonolignans obtained can be now used as authentic standards for in vivo metabolic experiments and for further research on their biological activity