Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 191-192.

Orientador: Paulo Mazzafera / Dissertação (mestrado) - Universidade Estadual de Campinas, Insituto de Biologia / Made available in DSpace on 2018-08-12T18:35:16Z (GMT). No. of bitstreams: 1 Andreazza_NathaliaLuiza_M.pdf: 3988387 bytes, checksum: 14f12a095b5cbb5989b090f57b961f01 (MD5) Previous issue date: 2009 / Resumo: A pilocarpina é um alcalóide imidazólico, que possui como única fonte natural spécies do gênero Pilocarpus. Este alcalóide é utilizado no tratamento de glaucoma e xerostomia. O elevado custo de folhas de Pilocarpus microphyllus no mercado internacional e conseqüente extrativismo predatório resultaram na sua inclusão na lista de espécies em extinção do IBAMA. Na busca de fontes alternativas do alcalóide conseguiu-se demonstrar que suspensões celulares desta espécie podem ser um modelo para produção e estudo da biossíntese e do transporte de pilocarpina, uma vez que produz os mesmos alcalóides encontrados nas folhas. A extração de pilocarpina a partir do meio de cultura poderá minimizar a quantidade de solventes altamente poluentes utilizados na extração deste alcalóide a partir das células, assim como, reduzir a contaminação por outros constituintes celulares. Neste contexto o presente trabalho teve como objetivo inicial determinar o local e limite de acúmulo intracelular de pilocarpina e verificar se o fornecimento externo de altas doses do alcalóide causaria toxidez às células produtoras e não produtoras de pilocarpina. Em seguida, caracterizar a absorção do alcalóide pelas células submetidas a diferentes valores de pH do meio de cultura e também identificar o mecanismo de transporte do alcalóide nas suspensões procurando definir qual é a proteína de membrana responsável pelo transporte de pilocarpina entre células e meio de cultura através do uso de inibidores de transportadores da família das ATPases e ATP-binding cassete proteins (ABC). Os testes histoquímicos e o ensaio de fracionamento celular, apesar de não conclusivos, indicaram o acúmulo de pilocarpina no vacúolo, ainda que a fração correspondente a essa organela venha misturada com o conteúdo do citoplasma. As informações sobre a localização subcelular em adição aos dados de toxicidade mostraram que pilocarpina apresenta forte citotoxicidade a cultura de plantas que não apresentam sua via de biossíntese. Culturas de P. microphyllus produtoras de pilocarpina apresentaram uma clara tolerância às altas doses do alcalóide (crescimento semelhante ao controle), mesmo que não produzindo o alcalóide em altas quantidades. Isto sugere a existência de um mecanismo de detoxificação espécifico-específico nas células aqui estudadas, que evitam a toxicidade de seus alcalóides (pilocarpina e pilosina) armazenando-os no vacúolo. Nos ensaios de absorção do alcalóide em diferentes valores de pH, observou-se que quanto maior o pH, menor a absorção do alcalóide. Nos ensaios com os inibidores de proteínas transportadoras de membrana verificou-se que as menores taxas de inibição na absorção e liberação provocadas por inibidores específicos de ATPases, a bafilomicina e pelo NH4Cl, não descartam a participação destas proteínas, mas podem indicam uma menor participação, visto que a inibição provocada pela azida sódica, também um inibidor de ATPases, foi muito intensa. Contudo, os resultados de absorção e liberação de pilocarpina mostraram intensa inibição na presença dos inibidores de ABCs o que aponta para um transporte de pilocarpina mediado por esta família de proteínas, tanto para fora como para dentro da célula. Por fim, os ensaios de cinética apontam para uma inibição do tipo competitiva gerada pelos dois inibidores utilizados, sendo que os menores valores da Constante de Inibição (Ki), encontrados para a nifedipina indicam que este composto possue uma ação inibitória mais intensa que o vanadato de sódio. / Abstract: Leaves of species from Pilocarpus genus are the only known source of pilocarpine, an imidazole alkaloid, which has been used for the treatment of glaucoma and xerostomy. Because the leaves of jaborandi are collected from plants living in the wild and the high price of pilocarpine in the international market, jaborandi was included in the endangered species list of IBAMA. Looking for alternative sources of this alkaloid, it has been shown that cell suspension cultures of Pilocarpus microphyllus can be a model to study the production of pilocarpine as well as a model to study its biosynthesis and metabolism, as it produces the same alkaloids that are found in leaves. Previous studies showed that high concentrations of nitrogen and the medium pH resulted in higher production and release of pilocarpine to the medium culture. Therefore, the objective of this study was to define the cell intracellular accumulation of pilocarpine and verify if exogenous by supplied pilocarpine to jaborandi cell suspensions is toxic to the cells. Moreover, the absorption of pilocarpine by cells treated with exogenous by supplied pilocarpine at different medium pH, as well as, the alkaloid transport mechanism through the cell membrane, using inhibitors of the protein families ATPases and ATP-Binding Cassette, were studied. The histochemical tests and the cell fractionation assays showed the accumulation of pilocarpine in the vacuole. This, together with the results of experiments that showed that pilocarpine was not toxic to jaborandi cells, suggests that vacuolar transport may be one of the mechanisms for the detoxification of pilocarpine in this species. In the absorption assays with different medium pH, the higher the pH, the lower absorption of pilocarpine by the cells. Bafilomicin and NH4Cl, which are ATPase inhibitors, were the least effective inhibitors among all the inhibitors tested for absorption and release of pilocarpine. This result does not discard the participation of these proteins in the process but indicate that they are less important, in view of the fact that inhibition by sodium azide which affects both ABC and ATPases, was very effective. The results on absorption and release of pilocarpine by the jaborandi cells showed strong inhibition by specific ABC inhibitors, which indicates an important participation of this protein family in the transport of the alkaloid through the cell membranes. Kinetics assays showed that inhibition was a reversible competitive type in the presence of nifedipine and sodium vanadate. The lowest Inhibition Constant (Ki) was observed for nifepidine. / Mestrado / Mestre em Biologia Vegetal

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types. / Science, Faculty of / Botany, Department of / Graduate

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences University of Vienna, Faculty of Life Sciences, Department of Pharmacognosy Candidate: Lenka Kovářová Supervisor: Pharmdr. Miroslav Kovařík, Ph.D. Consultant: Dr. Angela Ladurner Title of the diploma thesis: Characterization of a synthetic leoligin derivative, with agonistic FXR and enhancing macrophage cholesterol efflux activity Atherosclerosis is a pathologic multifactorial process triggering the development of cardiovascular diseases, which are the leading causes of death in the western world. The initial phase of atherosclerosis is characterized by the accumulation of lipid particles, mainly low-density lipoproteins (LDL) and macrophage-derived foam cells in large arteries, leading to the gradual thickening of the vessel wall. These progressive alterations elicit plaque formation, followed by rupture, thrombosis and finally can lead to a cardiovascular event. Reverse cholesterol transport is an important preventive mechanism, which ensures removal of excessive atherogenic lipoproteins from macrophages. This efflux is facilitated by ATP binding cassette transporters, mainly ABCA1 and ABCG1 and in part by scavenger receptor B1 (SR-B1). Several nuclear receptors, including PPARγ, LXRα and LXRβ...

The heterodimeric ATP-binding cassette (ABC) transporter, ABCG5/G8, is responsible for direct secretion of cholesterol and dietary sterols into the gut lumen and the bile. Inactivating mutations of ABCG5/G8 cause sitosterolemia, a rare autosomal recessive disease characterized by the accumulation of plant sterols in plasma, hypercholesterolemia and development of premature coronary heart disease. Functional and structural characterization of ABCG5/G8 is necessary to understand its mechanism and how the genetic defects impact its function. In this thesis, I expressed seventeen constructs of various disease-causing or catalytically deficient missense mutations in Pichia pastoris yeast. This establishes reagents for in vitro functional and structural studies. Secondly, I focused on two disease mutants (ABCG5-E146Q and ABCG8-R543S) and a sterol binding mutation (ABCG5-A540F) and established large-scale purification of these mutants. Using a cholesterol hemisuccinate (CHS)-dependent ATPase assay, I determined ATP hydrolysis by these three mutants and analyze their kinetic parameters. All missense mutants showed a significantly impaired ATPase activity, but the ability of ATP binding appeared unchanged between the WT and the mutants. This work demonstrates an intimate structure-function relationship in ABCG5/G8 and sheds some light on the mechanistic details of this important cholesterol-regulating ABC transporter.

Background The Breast Cancer Resistance Protein (BCRP) drug transporter is important for drug disposition and plays a critical role in regulating drug entry into the brain. Its substrate spectrum overlaps with substrates of Multi Drug Resistance Protein 1 (MDR1, P-gp), which influences and complicates the interpretation of data on drug distribution into tissues (e.g. brain). Distinguishing BCRP mediated transport from the transport by the MDR1 is often problematic. However, with new in vitro tools, this is now possible. In this project, two drug compounds, i.e. Dantrolene and Ritonavir, were investigated using these new in vitro models. The results from the experimental in vitro assay were matched with molecular dynamics (MD) simulations. Using coarse-grained (CG) simulations, a model of the BCRP transporter in a lipid bilayer was built, this model is based on the human BCRP structure revealed by Taylor et al (2017). Simulations were run for Dantrolene (a known substrate of BCRP) independently three times, and another with Ritonavir (a non-substrate) three times. Aim To determine substrate specificity for the BCRP transporter for two compounds, and to construct a CG model of BCRP transporter to see whether in silico methods can be used as an alternative for assessing substrate specificity.  Methods Madin-Darby canine kidney (MDCK) II cell line with no endogenous canine MDR1 (cMDR1) expression (MDCKcMDR1-KO), overexpressing human MDR1 (hMDR1) (MDCK-hMDR1cMDR1-KO) and stable expression of human BCRP (hBCRP) (MDCK-hBCRPcMDR1-KO) cells were cultured and used in Transwell experiments. Samples were analyzed using LC-MS/MS to determine the substrate concentrations. Apparent permeability and efflux ratio was calculated and evaluated.  MD simulations used the Martini 3 CG force field, and were run with Gromacs (version 2020.4). Tools including MODELLER, INSANE and others were used to construct the initial model (Webster, 2000; Wassenaar et al., 2015), for parameterization of substrate and non-substrate molecules. And visual inspection was done with the visual molecular dynamics (VMD) program and PyMOL. Results In vitro transport experiment confirmed that Dantrolene is a BCRP specific substrate, and Ritonavir is MDR1 specific substrate. Following simulations of these two compounds, Dantrolene is observed to stay in the transmembrane domains (TMD) for a certain period (on average several hundreds of nanoseconds), while Ritonavir is not found to bind in the TMD, which provides a proof of concept for future studies.

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McMaster University MASTER OF SCIENCE (2019) Hamilton, Ontario (Medical Sciences) TITLE: Modulation of cyclic adenosine monophosphate for potentiation of long-acting β2-agonist and glucocorticoids in human airway epithelial cells AUTHOR: Yechan Kim, B.HSc. (McMaster University) SUPERVISOR: Dr. Jeremy Alexander Hirota NUMBER OF PAGES: xiv, 81 / In Canada, asthma is the third most common chronic disease resulting in 250 premature deaths annually and related healthcare expenses exceeding $2.1 billion/year. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Despite an advanced understanding on how to treat and manage the symptoms of asthma, current therapy is sub-optimal in 35-50% of moderate-severe asthmatics around the world resulting in lung inflammation, persistent impairment of lung function, and increased risk of mortality. Combination of long-acting β2 agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma. Increasing intracellular cyclic adenosine monophosphate (cAMP) beyond existing combination LABA/GCS are likely to be beneficial for the management of difficult to control asthmatics that are hypo-responsive to mainstay therapy. In human airway epithelial cells (HAEC), cAMP is either exported by transporters or broken down by enzymes, such as phosphodiesterase 4 (PDE4). We have demonstrated that HAEC express ATP Binding Cassette Transporter C4 (ABCC4), an extracellular cAMP transporter. We also show that ABCC4 and PDE4 inhibition can potentiate LABA/GCS anti-inflammatory responses in a human epithelial cell line in a cAMP-dependent mechanism validating the pursuit of novel ABCC4 inhibitors as a cAMP elevating agent for asthma. / Thesis / Master of Science in Medical Sciences (MSMS) / Asthma is a common chronic lung disease characterized by narrow and inflamed airways that cause breathing difficulties. Current management includes the combination of bronchodilators, to relax the airway, and steroids, to decrease inflammation. Unfortunately, this combination therapy is suboptimal in 35-50% of users, increasing the risk of asthma attacks, hospitalization rate, and health care costs. Recently, there have been studies theorizing that we can improve the therapy’s ability to decrease inflammation by increasing cAMP, an important molecule for biological activities. We tested this claim by blocking the breakdown and export of cAMP to increase its levels and measured inflammatory cytokines, molecules that direct the action of immune cells. Our results show that in a model of viral infection, administering the combination therapy while increasing cAMP levels can further decrease inflammatory cytokines prompting further investigation for its potential implication in the clinic.