Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Eva Troppová Supervisor: PharmDr. Marie Vopršalová, CSc. Specialized supervisor: Prof. Dr. Antje Gohla Title of diploma thesis: Establishment of screening methods to find new regulators of the activity of phosphoglycolate phosphatase This work deals with the siRNA-based genomic screening for the modification of phosphoglycolate phosphatase (PGP) activity. 235 proteins were affected by transient transfection of siRNAs in vitro. Each siRNA was used in duplicates and the control was carried out by two control siRNAs. After downregulation of protein by siRNA PGP activity was evaluated, whether any modifications of PGP activity have occurred. PGP was the main research target. The main goal of this study before the screening was to set up a method, to create a reliable protocol. The whole study was 96 plate well. It was necessary to find the right conditions to measure PGP activity in two cell types (HEK AD 293 and Hep G2). Subsequently, optimal conditions were set up to influence expression of the protein. The method was optimalized using PGP siRNAs and 2 types of transfection reagents were tested. During our study the following methods were used: PGP activity assay, Bicinchoninic acid...

Abstract  Lack of clinical relevance is assumed to be the main reason behind the high failure rate of medical drugs in the very initial phases of clinical trials. Clinical relevance is difficult to achieve with current tools as they lack the biological and physiological cues found in vivo. Microfluidics, the knowledge of fluid manipulation in small channels, has proven to be a promising science to bridge the gap between the current in vitro and the real in vivo features. In this thesis, a scaffold for the growth of spheroids inside a microfluidic device for potential drug screening was developed. Firstly, the surface of a microfluidic device was coated with the polymers cellulose nanofibrils, polyallylamine hydrochloride, and polyethyleneimine using the Layer-by-Layer technique to achieve an even surface coverage. Here, different chip designs, polymer concentrations, and pressure directions were tested. It was decided that using a negative pressure direction with a polymer concentration of 50 mg/L in a chip design with micropillars was optimal and these conditions were then used for testing the spheroid formation. Secondly, spheroids were grown inside the microfluidic channels using different coatings: the previously mentioned polymer buildup, one non-coated channel, and one coated with attachment factor proteins. These three surface conditions were compared and it was shown that the polymer-based surface cover was indeed superior as a scaffold as it encouraged and promoted cell growth in the spheroid formation of liver cancer cells from the HepG2 cell line. Further development of this cellulose nanofibrils-coated microfluidic device displays a promising future for functioning as an in vitro 3D cell culture model that better mimics the close-to-cell microenvironments by imitating cell proliferation, cell-to-cell, and cell-to-extracellular matrix interactions. / Sammanfattning Den främsta orsaken bakom den höga antal misslyckade kliniska läkemedelsprövningar i de initiala faserna antas bero på brist på klinisk relevans. Klinisk relevans är mycket svår att uppnå med dagens verktyg då de saknar de biologiska och fysiologiska förhållandena som återfinns in vivo. Mikrofluidik, kunskapen om vätskemanipulation i små kanaler har visat sig vara lovande vetenskap för att överbrygga klyftan mellan de nuvarande in vitro och de faktiska in vivo funktionerna. I detta arbete utvecklades en matris för sfäroider att växa på inuti en mikrofluidisk kanal för att potentiellt användas till läkemedelsscreening. Först användes Layer-by-Layer teknologi för att jämnt betäckta ytan inuti en mikrofluidisk kanal med polymererna cellulosananofibriller, polyallylamin hydroklorid samt polyetylenimin. Här testades olika designer på mikrofluidiska chip, polymerkoncentrationer samt tryckriktningar. Utifrån detta gick det att fastställa att negativt tryck med en polymerkoncentration på 50 mg/L i en chippdesign med mikropelare var optimal för en jämn ytbetäckning och dessa förhållanden användes sedan för att pröva sfäroidernas tillväxt. Härnäst testades därmed sfäroidernas tillväxt inuti mikrofluidiska kanaler under tre olika förhållanden: ett med polymerbetäckningen, ett utan betäckning och ett då ytan var täckt med proteiner med fästfaktorer. Dessa tre förhållanden jämfördes sedan med varandra och således gick det att konstatera att den polymerbaseradebetäckningen fungerade överlägset som matris för tillväxt av HepG2 lever cancer cell sfäroider eftersom den tycks främja dess tillväxt och bildning. Det pekar mot att ytterligare utveckling av denna cellulostäckta yta skulle innebära en lovande modell för in vitro 3D cellodling som bättre efterliknar den cellulära mikromiljön genom att imitera cellproliferation, interaktioner celler emellan samt mellan cell och extracellulär matrisen.

Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-04-17T11:40:30Z No. of bitstreams: 1 KELLY_GOULART_LIMA_TES.pdf: 13016256 bytes, checksum: b2a78492cbc6e285501d0eed5538a8ee (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-05-04T16:48:04Z (GMT) No. of bitstreams: 1 KELLY_GOULART_LIMA_TES.pdf: 13016256 bytes, checksum: b2a78492cbc6e285501d0eed5538a8ee (MD5) / Made available in DSpace on 2018-05-04T16:56:22Z (GMT). No. of bitstreams: 1 KELLY_GOULART_LIMA_TES.pdf: 13016256 bytes, checksum: b2a78492cbc6e285501d0eed5538a8ee (MD5) Previous issue date: 2018-03-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Octyl gallate (OG) is an antioxidant used in the food, cosmetic and medications industries that has shown antitumor effect on cell lineage of leukemia, melanoma and B cell lymphoma, as well as on animal model of pulmonary metastasis. Hepatocellular carcinoma (HC) is the main primary liver cancer affecting the world's population. Although surgical resection, ablation and liver transplantation are curative options, few patients are eligible for these therapies. The application of these therapeutic options is indicated only in the early stages of the disease and unfortunately most patients are diagnosed at advanced stage. In addition, there are reports in the literature of resistance to the only drug approved for systemic treatment, sorafenibe. In this study, we investigated the effect of OG on cell proliferation and lipidic metabolism in hepatocellular carcinoma HepG2 cells. Moreover, we developed a protocol for the quantitative evaluation of lipid droplets. We report, for the first time, that treatment with OG for 24 h inhibited HepG2 cell growth by decreasing mitochondrial activity and mass, which led to the reduction of ATP levels. This reduction in the energy supply triggered a decrease in Ki67 protein expression, leading to cycle arrest in S phase. In addition, the use of two treatments with OG with interval 24 hours induced loss of mitochondrial functionality and apoptosis without inducing resistance. These results showed that OG targets the mitochondria and is a candidate for new research on therapies for CH. We also report, for the first time, the effect of OG on lipidic metabolism, since our results showed that OG was able to increase the amount of lipids, triglyceride levels and the area of lipid droplets without involving the mTOR/SREBP-1c signaling pathway or modification of PPAR-? and PPAR-? gene expression. As the ability of OG to inhibit mitochondrial activity and induce apoptosis is known, it is strongly suggested that reduction of mitochondrial fatty acid b-oxidation is involved in the OG mechanism in the accumulation of lipids. We also describe, for the first time, a protocol for the quantitative evaluation of lipid droplets using confocal laser scanning microscopy that compared to conventional fluorescence microscopy, provided great gain in the quality of the images. / O galato de octila (GO) ? um antioxidante utilizado na ind?stria de alimentos, cosm?ticos e de medicamentos que tem mostrado efeito antitumoral em linhagem celular de leucemia, melanoma e linfoma de c?lulas B, assim como em modelo animal de met?stase pulmonar. O Carcinoma hepatocelular (CH) ? o principal c?ncer prim?rio hep?tico que afeta a popula??o mundial. Embora a ressec??o cir?rgica, a abla??o e o transplante hep?tico sejam considerados terapias curativas, poucos pacientes s?o eleg?veis. A aplica??o dessas op??es terap?uticas ? indicada somente para est?gios iniciais da doen?a e infelizmente a maioria dos pacientes ? diagnosticado em est?gio avan?ado. Al?m disso, existem relatos na literatura de resist?ncia ? ?nica droga aprovada para tratamento sist?mico, o sorafenibe. Nesse estudo, n?s investigamos o efeito do GO sobre a prolifera??o celular e metabolismo lip?dico nas c?lulas de carcinoma hepatocelular HepG2. Al?m disso, desenvolvemos um protocolo para avalia??o quantitativa de gotas lip?dicas. N?s reportamos, pela primeira vez, que o tratamento com GO por 24 horas inibiu a prolifera??o das c?lulas HepG2 por reduzir a atividade e massa mitocondrial, que levou ? redu??o dos n?veis de ATP. Essa redu??o no fornecimento de energia desencadeou a diminui??o na express?o da prote?na Ki67, levando ? parada do ciclo celular em fase S. Al?m disso, o uso de dois tratamentos com GO com intervalo de 24 horas induziu perda da funcionalidade mitocondrial e apoptose, sem induzir resist?ncia. Esses resultados mostraram que o GO tem como alvo a mitoc?ndria, sendo um candidato para novas pesquisas sobre terapias para o CH. Reportamos tamb?m, pela primeira vez, o efeito do GO sobre o metabolismo lip?dico, visto que nossos resultados mostraram que o composto foi capaz de aumentar a quantidade de lip?deos, os n?veis de triglicer?deos e a ?rea de gotas lip?dicas, sem envolver a via de sinaliza??o mTOR/SREBP-1c ou a modifica??o da express?o dos genes PPAR-? e PPAR-?. Uma vez que a capacidade do GO em inibir a atividade mitocondrial e induzir a apoptose ? conhecida, ? fortemente sugerido que a redu??o da ?-oxida??o mitocondrial de ?cidos graxos esteja envolvida no mecanismo do GO no ac?mulo de lip?deos. Descrevemos tamb?m, pela primeira vez, um protocolo para avalia??o quantitativa de gotas lip?dicas usando microscopia confocal de varredura a laser que comparado ? microscopia de fluoresc?ncia convencional, proporcionou grande ganho na qualidade das imagens.

Secreted proteins play important roles in many cellular functions and molecular processes. Because secreted proteins potentially enter the blood stream, they can serve as valuable measures of health and disease useful for disease diagnosis and prognosis, therapeutic target identification, and patient stratification in personalized medicine. Consequently, significant interest exists in secreted protein analysis within complex biospecimens, particularly blood but significant bioanalytical challenges including the wide protein dynamic range >10 orders of magnitude remain. The cellular secretome therefore represents a viable alternative to direct biomarker discovery in biofluids. Finally, cellular systems are amenable to labeling for the production of intact stable isotope labeled (SIL) proteins that can be used as global internal standards for quantitative proteomics. In this dissertation, two secretome-focused studies were undertaken. The first study involving candidate biomarker discovery in radiation-induced autophagy utilized the p53-null and inducible H1299 non-small cell lung cancer (NSCLC) secretome. The study identified 364 secreted proteins that were mainly associated with exosomes (N=224) and chaperone activity (N=21). CHGB and SCG2 were identified as potential population-based biomarkers (for patient stratification) due to their consistent overexpression in p53-null H1299 cell secretomes compared to p53-wt cells before and after radiation. FAM3C, CANX, EIF5A, GPI, and TXNRD1 were identified as candidate biomarkers for patient prognosis following radiotherapy due to their differential expression only in response to radiation treatment. In the second study, a comprehensive glycoproteomics characterization of the SILAC-labeled HepG2 secretome was undertaken. 1635 SIL proteins, 492 of which were major plasma proteins including 192 cancer biomarkers were identified with high sequence coverage spanning six orders of magnitude. EDTA plasma spiked with the SIL secretomes yielded 63 proteins that were quantified with H/L ratios in all samples out of 1405 total proteins identified. Additionally, LC-MS/MS analysis of the Con A and WGA enriched 72h secretome:plasma sample afforded an opportunity to clearly distinguish between glycoproteins in plasma and the HepG2 secretome that share/differ in N-glycan structures. Collectively, the two studies reveal the suitability of the H1299 cancer cell secretome as an experimental model for biomarker studies and support the HepG2 secretome as a viable platform for producing SIL glycoproteins.

The current dietary recommendations for optimal health are designed to increase our intake of two bioactive omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), abundant naturally in fatty fish such as salmon. Health authorities recommend that the general population consume two to three fatty fish meals per week (1) for optimal health and for the prevention of cardiovascular disease. However, some modern Western societies consume only modest amounts of fish and seafood (2;3). Land based vegetable oils may provide an alternative to meet these needs. Linseed and canola oils are rich in alpha-linolenic acid (ALA, 18:3n-3) (4). ALA can be converted endogenously to EPA and DHA and suggests that increasing the dietary intake of ALA may increase the conversion and accumulation of DHA in tissues and plasma. However, elevated dietary intakes of ALA in animals and humans results in an increased level of EPA in tissues yet there is little or no change in the level of DHA (5-7). The current consensus is that the synthesis of DHA from ALA in humans is limited yet the mechanisms involved in regulating the accumulation of DHA in tissues are poorly understood. The reputed rate-limiting enzyme in the conversion of fatty acids is delta 6 desaturase (D6D). ALA is a substrate for D6D and undergoes a series of desaturation and elongation reactions to yield n-3 long chain polyunsaturated fatty acids (LCPUFA). The final step in the synthesis of DHA from ALA involves translocation of its immediate fatty acid precursor, 24:6n-3 from the endoplasmic reticulum to the peroxisome to be partially beta-oxidised to yield DHA. The involvement of multiple enzymes in the desaturation-elongation pathway, and the integration of other pathways, such as phospholipid biosynthesis, suggests there are various steps that may regulate the accumulation of DHA in cell membranes. This thesis aimed to examine the possible regulatory steps in the conversion of fatty acids to LCPUFA, particularly in the synthesis of DHA from n-3 fatty acid precursors. The human hepatoma cell line, HepG2, was used as an in vitro cell system to examine the accumulation of individual fatty acids and their metabolites in isolation from other competing fatty acid substrates. The accumulation of linoleic acid (LA, 18:2n-6) and ALA in HepG2 cell phospholipids following supplementation with increasing concentrations of each respective fatty acid correlated with that described in vivo, as was the accumulation of their conversion products. The accumulation of DHA in cells supplemented with ALA reached a plateau at concentrations above 5 micro g/ml and paralleled the accumulation of 24:6n-3 in cell phospholipids, suggesting that the delta 6 desaturation of 24:6n-3 was prevented by increasing concentrations of ALA, thereby limiting the accumulation of DHA. The accumulation of DHA in cells supplemented with eicosapentaenoic acid (EPA, 20:5n-3) or docosapentaenoic acid (DPA, 22:5n-3) was significantly greater than the level of DHA that accumulated in cells supplemented with ALA. However, regardless of substrate, the level of DHA in cell membranes reached a plateau at substrate concentrations above 5 micro g/ml. This thesis further aimed to examine the effect of fatty acid supplementation on the mRNA expression of D6D in HepG2 cells. The expression and activity of D6D mRNA is subject to nutritional and hormonal regulation. The mRNA expression of D6D in HepG2 cells following supplementation with oleic acid (OA, 18:1n-9), LA, ALA, arachidonic acid (AA, 20:4n-6) or EPA was examined by real time RT PCR. The expression of D6D mRNA was reduced by up to 50% in cells supplemented with OA, LA, ALA , AA or EPA compared with control cells and suggests that fatty acids modulate the expression of the key enzyme involved in the conversion of fatty acids. The effect of fatty acid co-supplementation on the fatty acid composition of HepG2 cell phospholipids was also examined in an attempt to gain insights into the role of D6D and the enzymes involved in peroxisomal beta-oxidation on the accumulation of DHA from n-3 fatty acid precursors. The reduction in the accumulation of DHA in cells co-supplemented with DPA and docosatetraenoic acid (DTA, 22:4n-6) was greater than in cells co-supplemented with DPA and LA, suggesting that peroxisomal beta-oxidation may have a greater role in determining the accumulation of DHA from DPA than the activity of D6D. Further investigation should be directed towards understanding the role that peroxisomal beta-oxidation may play in the synthesis of DHA from precursor fatty acids. The fatty acid composition of cell membranes in vivo is a result of several physiological processes including dietary intake, phospholipids biosynthesis and fatty acid conversion as well as catabolic processes. This thesis demonstrates that a greater understanding of the regulation of the conversion of fatty acids will help to define dietary approaches that enhance the synthesis of n-3 LCPUFA from n-3 fatty acid precursors to lead to improved outcomes for health.

Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

The overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated with metabolic dysregulation and insulin resistance. Because mitochondria generate reactive oxygen species (ROS), we monitored changes to the adipocyte mitochondrial proteome during differentiation and enlargement. We labeled mitochondrial extracts from 3T3-L1 cells that were 0, 4, 7, 10, 14, and 18 days post differentiation with iTRAQ, followed by MS based identification. We found citric acid cycle proteins such as pyruvate carboxylase, citrate synthase, as well as beta-oxidation enzymes; cartinine acyl transferase and long-chain enoyl-CoA hydratase up-regulated from 7 through 18 days post differentiation onset. These data indicate TCA up-regulation for enhanced metabolic and citrate output necessary for lipid synthesis in adipocytes. Paradoxically, the data also show the simultaneous increase in the fatty acid oxidation, indicating a metabolic overdrive state. Biochemical assays showing peaks in ATP and ROS generation in 3 day old adipocytes provide further evidence of this overdrive state. A second peak in ROS generation occurred in 10 day old adipocytes; concurrent ATP generation reduced to near pre-adipocyte levels and this may indicate a metabolic shift that may be responsible for increased oxidative stress in hypertrophic adipocytes. We developed a doxycycline inducible CYP2E1 expressing HepG2 cell line using the pTet-On/pRevTRE expression system to allow greater control and sensitivity in the generation CYP2E1 mediated oxidative stress. Our cell line (RD12) demonstrated stability and tight expression control. After induction, RD12 cells showed 30 percent higher CYP2E1 activity when compared to the constitutive E47 cell line. RD12 cells showed 30 percent greater toxicity than E47 cells and 25 percent less free glutathione when exposed to 20 mM acetaminophen, indicating RD12 cells are more sensitive to the effects reactive intermediates and oxidative stress generated by CYP2E1. We conducted a survey of the toxicity of dietary fatty acids (oleic, linoleic, and palmitic) on HepG2 cells to determine fatty acid doses that induced metabolic changes, but did not cause excessive cell death. The dose of 0.20 mM linoleic and palmitic acid for 48 hours produced low toxicity, but oleic acid actually produced lower toxicity than untreated cells. After exposure cells were treated with a pro-oxidant to determine which fatty acid increased the susceptibility to protein carbonylation. The carbonylated protein isolation procedure indicated the palmitic acid may induce more carbonylation than oleic acid, but greater efficiency in the isolation procedure is required for a confidant determination.

Hypercholesterolemia, a condition of high cholesterol levels in the circulation, poses a major risk for developing cardiovascular disease, such as atherosclerosis. A common method of reducing plasma cholesterol levels relies on the administration of drugs that limit cholesterol synthesis or uptake, many of which have undesirable side effects. Thus, some patients are turning to an alternative treatment, namely natural health products. Natural health products are often equally or even more effective at treating illness than synthetic drugs and may produce fewer side effects. The goal of this study was to identify a natural health product that regulates hepatic cholesterol synthesis by inhibiting HMG-CoA reductase, the enzyme which catalyzes the rate-limiting step of the cholesterol synthesis pathway. Several natural compounds were screened using the human hepatoma cell line HepG2. One compound, berberine, showed great potential as a regulator of cholesterol synthesis and so became the subject of this investigation. Berberine inhibited HMG-CoA reductase activity and decreased cellular accumulation of cholesterol. Berberine was shown to regulate HMG-CoA reductase through activation of metabolic regulator AMP-activated protein kinase, which modifies HMG-CoA reductase post-translationally and thereby decreases its activity. In conclusion, this study demonstrates that the natural health product berberine decreases cholesterol synthesis by activating a cellular signalling pathway to bring about post-translational modification of HMG-CoA reductase, and in doing so, inhibits this enzyme. This novel mechanism supports berberine’s potential for a cholesterol-lowering therapy and its role in reducing the risk for cardiovascular disease.

Hypercholesterolemia, a condition of high cholesterol levels in the circulation, poses a major risk for developing cardiovascular disease, such as atherosclerosis. A common method of reducing plasma cholesterol levels relies on the administration of drugs that limit cholesterol synthesis or uptake, many of which have undesirable side effects. Thus, some patients are turning to an alternative treatment, namely natural health products. Natural health products are often equally or even more effective at treating illness than synthetic drugs and may produce fewer side effects. The goal of this study was to identify a natural health product that regulates hepatic cholesterol synthesis by inhibiting HMG-CoA reductase, the enzyme which catalyzes the rate-limiting step of the cholesterol synthesis pathway. Several natural compounds were screened using the human hepatoma cell line HepG2. One compound, berberine, showed great potential as a regulator of cholesterol synthesis and so became the subject of this investigation. Berberine inhibited HMG-CoA reductase activity and decreased cellular accumulation of cholesterol. Berberine was shown to regulate HMG-CoA reductase through activation of metabolic regulator AMP-activated protein kinase, which modifies HMG-CoA reductase post-translationally and thereby decreases its activity. In conclusion, this study demonstrates that the natural health product berberine decreases cholesterol synthesis by activating a cellular signalling pathway to bring about post-translational modification of HMG-CoA reductase, and in doing so, inhibits this enzyme. This novel mechanism supports berberine’s potential for a cholesterol-lowering therapy and its role in reducing the risk for cardiovascular disease.