4 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Dimitrios Vagiannis Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Evaluation of antiproliferative effect of selected tyrosine kinase inhibitors in MDCKII cell lines Tyrosine kinases are important enzymes regulating crucial cellular processes including differentiation, proliferation, apoptosis, transcription, metabolism, and intercellular communication. Deregulation of these enzymes is the cause of various types of cancers. The blockade of their function by tyrosine kinase inhibitors (TKis) is considered a promising approach especially in antitumor pharmacotherapy. ATP-binding cassette (ABC) drug efflux transporters are a family of transmembrane proteins that pump a variety of structurally unrelated compounds out of the cell in an energy-dependent manner. They play an important role in pharmacokinetics (affect absorption, distribution, elimination) and, at the same time, can negatively influence efficacy of chemotherapy (participate in multidrug resistance phenomenon). In our research, we evaluated antiproliferative properties of four selected TKis, namely alectinib, brivanib, osimertinib and selumetinib, in MDCKII cell lines (parent one and those transduced with human...

Reducing the rates of greenhouse gas (GHG) emissions is critical in combatting global climate change. Carbon dioxide (CO2) and methane (CH4) are the two most important carbon-based GHGs, for their atmospheric warming potential. Wetlands such as the Florida Everglades play major roles in the global carbon cycle, as varying hydrologic conditions lead to differential production rates of these two GHGs. This study measured CO2 and CH4 emissions in a re-created Everglades ridge-and-slough wetland, where water levels were controlled to reflect natural flood patterns. As expected, lower elevations were flooded longer and produced more CH4, while higher elevations produced more CO2. Since CH4 has a relatively high global warming potential, CO2 production would need to be 70 times that of CH4, to balance their GHG output. The average ratio of CO2 to CH4 across elevations was 22.0 (mol:mol), indicating that future water management within wetlands should consider GHG production potential.

P. aeruginosa e espécies de Acinetobacter são causas comuns de diversas infecções em pacientes hospitalizados, principalmente nos internados em centros de tratamento intensivo. Além disso, esses microrganismos se destacam por apresentarem resistência, intrínseca e adquirida, a várias classes de antibióticos, conferindo à bactéria fenótipos de multirresistência e panresistência. O objetivo deste estudo foi avaliar a participação de integrons (elementos genéticos que carreiam genes de resistência), de genes codificadores de metalo--lactamases, da perda de porinas (canais protéicos da membrana externa), e da atividade de efluxo aumentada, como determinantes do fenótipo de multirresistência e panresistência. Foram estudadas 147 P. aeruginosa e 57 Acinetobacter spp. isolados de pacientes hospitalizados no Hospital Universitário da Universidade Federal de Juiz de Fora, no período de 2003 a 2006. O perfil de sensibilidade destes isolados foi determinado por disco de difusão e utilizado para classificá-las como multirresistentes (MDR) e não multirresistentes (n-MDR). A variabilidade clonal dos isolados foi investigada por PFGE. Os isolados pertencentes aos grupos MDR e n-MDR foram investigados quanto a presença de integrons de classe 1, 2 e 3, por PCR e análise de RFLP. Os cassetes gênicos contidos nestes integrons, assim como genes codificadores de carbapenemases (ex. IMP, VIM e SPM), foram detectados por PCR e identificados por seqüenciamento. Avaliação da expressão gênica de bombas de efluxo (mexB, mexY, mexD e adeB) e de porina (OprD) foi conduzida por real-time RT-PCR. Os dados apresentados para os isolados do grupo MDR foram comparados àqueles do grupo n-MDR e a associação entre os determinantes de resistência e o fenótipo MDR foi calculada estatisticamente. Fenótipo de multiresistência foi observado em 42,2% e 84,2% das P. aeruginosa e Acinetobacter spp. estudadas. Nenhum isolado bacteriano apresentou fenótipo panresistente. Em 65 (44,2%) dos isolados de P. aeruginosa, foram detectados integrons de classe 1. Esses elementos apresentaram relação estatisticamente significativa com fenótipos MDR em P. aeruginosa. Entretanto, a maioria desses integrons não carreava nenhum cassete gênico (43/65) ou continham apenas cassetes gênicos de resistência a aminoglicosídeos (19/65). Entre os isolados de Acinetobacter spp., 11 (17,5%) apresentaram integrons de classe 1 e 30 (47,6%) integrons de classe 2. Apenas os últimos foram estatisticamente associados com fenótipos MDR. A pesquisa de metalo--lactamase (MBL) revelou a produção de enzimas SPM em 24 isolados de P. aeruginosa. Os estudos de expresão gênica demonstraram que, entre os sistemas de efluxo mais relatados para P. aeruginosa, MexXY-OprM foi o que mostrou maior diferença entre o nível de expressão dos grupos MDR e n-MDR, sugerindo que este sistema de efluxo desempenha importante papel no fenótipo MDR. Diminuição, em média de 66,4%, da produçãode OprD também foi um padrão encontrado nos isolados MDRem relação aos n-MDR. Dois grupos clonais de P. aeruginosa e dois de Acinetobacter spp. foram predominantes e tiveram relação com presença de integrons, produção de SPM-1 e com fenótipo MDR. Portanto, esse fenótipo pode ser consequência de acúmulo de determinantes de resistência em clones específicos. / The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii.

Aim: The general aim of this research is development and evaluation of novel methods for predicting active transport over the human blood-brain-barrier (BBB), while this project specifically aims to i) review the literature and select suitable methods and substrates, ii) develop models for determining in vitro kinetic properties of selected compounds, analyze the in vitro data using the developed models and to use Maximum Transport Activity (MTA) approach (Karlgren et al., 2012), iii) perform Physiology Based Pharmacokinetic (PBPK) modelling and compare to in vivo literature data. Background: Drug permeation to the brain through blood circulation is primarily limited by blood-brain barrier (BBB), due to existence of tight junctions in endothelial cells of blood vessels as well as active efflux and influx transporters in the barrier. Toxicity and CNS related side effects can be caused by peripheral targeted drugs crossing BBB. Hence, prediction of BBB permeability and estimation of drug concentration in the brain tissue are challenging in drug discovery. To resolve this, estimating the human BBB permeability using improved in vitro and in silico predictive models can be a facilitator. Methods: In vitro data provided by the Drug Delivery research group was used to develop in vitro predictive models for BBB penetration of Verapamil, Risperidone, and Prazosin using R-studio 1.2.5. The MTA approach was adjusted for extrapolation of BBB in vitro transporter activity to in vivo condition. For PBPK modelling, we took advantage of PK-Sim® to simulate drug disposition and time profile of Risperidone in human and animal species. Results: It was shown that MDR1 is the major transporter for efflux transport of Prazosin and Risperidone in brain while both BCRP and MDR1 have similar impact on transport of Verapamil. Furthermore, it was presented in PBPK models that the predicted brain concentration of Risperidone increases in rat and nonhuman primate (NHP) when MDR1 And BCRP are knocked out while the brain concentration of Risperidone in dog is not affected by expression level of the efflux transporters. Conclusion: Both MDR1 and BCRP are contributing in efflux transport of Verapamil, Risperidone, and Prazosin across the BBB. Additionally, expression of the efflux transporters shown to have an impact on brain exposure of Risperidone in animal PBPK models.

Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...

Charles University Faculty of Pharmacy in Hradec Kralove Department of Pharmacology & Toxicology Student: Gabriela Burianova Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Flow-cytometric analysis of inhibitory effect of novel targeted drugs on the activity of ABC drug efflux transporters Cancer is the second leading cause of death. Cancer treatment often combines conventional chemotherapy, radiation therapy and surgery. More recent approach to treatment is the use of targeted cancer therapy with a greater specificity towards cancer cells. Development of resistance is a major obstacle in the success of chemotherapy. Multidrug resistance (MDR) can be acquired through various mechanisms e.g. overexpression of efflux transporters. ATP binding cassette (ABC) transporters represents a large family of transmembrane proteins that use ATP to pump molecules across the membrane. The three main ABC proteins related to MDR are: P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2). Use of ABC transporter inhibitors increases the amount of chemotherapeutical substrates accumulated within the cells. In this study we evaluated interactions of six synthetic small molecule inhibitors (alisertib, ensartinib, entrectinib, talazoparib,...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucia Dziaková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on interactions of PARP inhibitors with ABC drug efflux transporters. ATP-binding cassette (ABC) transporters are integral membrane proteins that use the energy obtained from ATP to carry transport of numerous endogenous substrances out of the cells, but attention is drawn primarily to the fact that they transfer also xenobiotics. Their overexpression in tumor tissue contributes to multidrug resistance (MDR), which in most cases leads to therapy failure. Poly(ADP-ribose)polymerase inhibitors (PARPi) represent a promising therapeutic approach in the treatment of cancers that exhibit defects in homologous recombination (HR). This work focuses on four selected PARPi (olaparib, rucaparib, niraparib, veliparib) and their interaction potential towards ABC drug efflux transporters (ABCB, ABCC1, ABCG2). In our work, we worked with MDCKII cells (parent, transduced by the transporters of interest) and utilized the principle of accumulation studies based on the measurement of fluorescence intensity of specific model substrates (hoechst33342, calcein AM, daunorubicin, mitoxantrone). We used established inhibitors of studied...

Tocopheryl Polyethylene Glycol Succinate 1000 (TPGS 1000) can inhibit P-glycoprotein (P-gp); TPGS 1000 was not originally designed to inhibit an efflux pump. Recent work from our laboratories demonstrated that TPGS activity has a rational PEG chain length dependency. In other recent work, inhibition mechanism was investigated and appears to be specific to the ATPase providing P-gp energy. Based on these observations, we commenced rational surface-active design. The current work summarizes new materials tested in a validated Caco-2 cell monolayer model; rhodamine 123 (10 μM) was used as the P-gp substrate. These results demonstrate that one may logically construct non-ionic surfactants with enhanced propensity to inhibit in vitro efflux. One new surfactant based inhibitor, Tocopheryl Polypropylene Glycol Succinate 1000 (TPPG 1000), approached cyclosporine (CsA) in its in vitro efflux inhibitory potency. Subsequently, TPPG 1000 was tested for its ability to enhance the bioavailability of raloxifene - an established P-gp substrate - in fasted male rats. Animals dosed with raloxifene and TPPG 1000 experienced an increase in raloxifene oral bioavailability versus a control group which received no inhibitor. These preliminary results demonstrate that one may prepare TPGS analogs that possess enhanced inhibitory potency in vitro and in vivo.

Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Júlia Jurčáková Supervisor: RNDr. Jakub Hofman PhD. Title of diploma thesis: The assessment of inhibitory effects of selected targeted anticancer drugs on the activity of ABC drug eflux trasporters. Lung cancer is the leading cause of death within oncological diseases. Non-small cell lung carcinoma (NSCLC) accounts for about 85% of all lung cancer, and its major subtypes include adenocarcinoma and squamous cell carcinoma. In addition to surgery, radiotherapy and chemotherapy, the use of targeted low-molecular substances, which target tumor cells with higher specificity, has recently been used in treatment. The two main causes of death in cancer patients are the formation of metastases and the development of multidrug resistance (MDR). This may also be caused by overexpression of the efflux transporters. ATP-binding cassette (ABC) transporters are groups of transmembrane pumps that use energy in the form of ATP to transfer a wide range of substrates. In particular, P-glycoprotein (ABCB1), breast cancer-resistance protein (ABCG2) and multidrug resistance-associated protein 1 (ABCC1) are associated with MDR. Inhibition of these transporters increases the amount of cytostatic substrate within the...