Uric acid is a main metabolite of purine degradation in humans and in higher primates. Its increased plasmatic level is called hyperuricemia and may be the cause of gout and many other similar diseases. Uricemia is controlled by many transporters, which are located in proximal tubule of human kidney. When some transporter have abnormal function, the physiological plasmatic level of uric acid may be impaired. In genome wide association study (GWAS) it was discovered that some hyperuricemia or gout patients have ABCG2 protein damaged. This protein carries out uric acid from epithelial cell to the urine. The goal of this diploma thesis is the determination of transport capacity of ABCG2 allelic variants found via GWAS (Institute of Rheumatology of 1st medical faculty UK in Prague) in vitro with Xenopus laevis oocyte expression system. Uric acid secretion was compared with wild type variant. Keywords: Uric acid, GWAS study, Xenopus laevis, membrane transport protein, ABCG2

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009.

Type II secretion system (T2SS) is one of the secretion systems found in gram-negative bacteria that provides transport of some bacterial proteins across the outer membrane. The passage through the membrane is mediated by a pore assembled from subunits called GspD or secretin. Together with three other components of T2SS, GspD was discovered in the genome of several protists including Naegleria gruberi, Andalucia godoyi, Reclinomonas americana, Neovahlkampfia damariscottae or in s species of genus Malawimonas. Previously it was found out that these proteins localize into the mitochondria. If found functional and with analogous topology to the bacterial system, the eukaryotic T2SS would represent unique mitochondrial protein export system. Secretin is essential subunit of T2SS which is not only the passive membrane channel, but also participates in the recognition of the substrate. Therefore, the research of the eukaryotic secretin could bring a valuable knowledge about the function of the mitochondrial T2SS. The experimental part of this thesis tries to characterize the eukaryotic secretin and it focuses on (i) the assembly of the secretin channel, in both, the bacteria and in the artificial membranes, (ii) the interactions of GspD with the other subunits of T2SS and (iii) the mechanism of import...

The glucose transport protein, GLUT1, is highly expressed in rapidly proliferating cells, including cancer cells, while decreased GLUT1 levels are found in diseases such as GLUT1 deficiency syndrome and Alzheimer’s. There is increased interest in developing GLUT1 inhibitors as novel anticancer therapeutics, and the discovery of compounds that directly stimulate GLUT1 function. This work investigates how small molecules stimulate and/or inhibit GLUT1-mediated glucose transport, either directly or through the AMPK pathway. Using sugar transport assays and docking analyses to explore Ligand–GLUT1 interactions and specificity of binding, we show that: 1) Ligands inhibit GLUT1 by competing with glucose for binding to the exofacial or endofacial sugar binding sites; 2) Subsaturating inhibitor concentrations stimulate sugar uptake; 3) Ligands inhibit GLUT1–, GLUT3– and GLUT4–mediated sugar uptake in HEK293 cells; and 4) Inclusion of a benzonitrile head group on endofacial GLUT1 inhibitors confers greater inhibitory potency. Furthermore, we investigated AMPK-regulated GLUT1 trafficking in cultured blood-brain barrier endothelial cells, and show that inhibition of GLUT1 internalization is not responsible for increased cell surface levels of GLUT1 observed with AMPK activation in these cells. This study provides a framework for screening candidate GLUT1 inhibitors for specificity, and for optimizing drug design and delivery. Our data on transport stimulation at low inhibitor concentrations support the idea that GLUT1 functions as a cooperative oligomer of allosteric alternating access subunits.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models were developed.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Coleoptera is an order of insects well known as beetles. Most coleopteran species are phytophagous insects and for this reason are essential to crop and storage pests such as the Tribolium castaneum. Lipid metabolism is vital for the biological functions of insects, playing a role in the generation of metabolic energy and other cellular processes. Fatty acid transport proteins (FATPs) play a crucial role in the transport of extracellular fatty acids to cells, have a conserved sequence between species and are involved in the synthesis of hormones and pheromones. Recent studies show that silencing the gene for FATPs through interfering RNAi techniques (RNAi) in insects affects fatty acid uptake and pheromone synthesis. This work aims to characterize proteins homologous to FATPs present in the genome of Tribolium castaneum, to evaluate the gene expression in tissues, developmental stages and insects treated with Orlistat and to evaluate the effect of FATP silencing on energy metabolism. Bioinformatics analyzes were performed with the amino acid sequences, and real-time PCR evaluated the gene expression. The effects of the drug Orlistat were evaluated through qPCR and analysis of nutritional index. The search for sequences in the T. castaneum genome revealed two sequences of proteins homologous to FATPs and bioinformatic analysis was performed. The study of the gene expression of FATPs by qPCR demonstrated more significant expression of the two genes in the fat body of larvae and many expressions in all stages of development of the insect, with higher expression in the pupa stage. The effects of Orlistat on the expression of FATPs evidenced the influence of diet composition on the regulation of the gene expression of these proteins. Gene silencing of TcasFATP was achieved, but no direct effects on the energetic dynamics of the larvae were observed since triacylglycerol levels, and β-oxidation rates were not affected. Thus, more detailed studies with the use of gene silencing will be necessary to characterize FATPs better and elucidate their role in insect energy metabolism. / FAPEAL - Fundação de Amparo à Pesquisa do Estado de Alagoas / Os coleópteros constituem uma ordem de insetos bastante conhecidos como besouros. A maioria das espécies de coleóptero são insetos fitófagos e por esta razão constituem importantes pragas de culturas e de armazenamento, como o Tribolium castaneum. O metabolismo de lipídeos é importante para as funções biológicas de insetos, exercendo papel na geração de energia metabólica e em outros processos celulares. As proteínas transportadoras de ácidos graxos (FATPs) exercem papel crucial no transporte de ácidos graxos extracelulares para as células, possuem sequência conservada entre as espécies e estão envolvidas na síntese de hormônios e feromônios. Estudos recentes mostram que o silenciamento do gene para FATPs através de técnicas de RNA de interferência (RNAi) em insetos afetam a absorção de ácidos graxos e a síntese de feromônio. Este trabalho tem como objetivo caracterizar proteínas homólogas à FATPs presentes no genoma de Tribolium castaneum, avaliar a expressão gênica nos tecidos, fases de desenvolvimento e em insetos tratados com Orlistate e avaliar o efeito do silenciamento de FATPs no metabolismo energético. Foram realizadas análises bioinformáticas com as sequências de aminoácidos e a avaliação da expressão gênica foi realizada por PCR em tempo real. Os efeitos do fármaco Orlistate foram avaliados através de qPCR e análise dos índices nutricionais. A busca de sequências no genoma do T. castaneum revelou duas sequências de proteínas homólogas à FATPs e a análise bioinformática foi realizada. O estudo da expressão gênica de FATPs por qPCR demonstrou maior expressão dos dois genes no corpo gorduroso de larvas e expressão considerável em todos os estágios de desenvolvimento do inseto, com maior expressão no estágio de pupa. Os efeitos do Orlistate na expressão das FATPs evidenciaram a influência da composição da dieta na regulação da expressão gênica dessas proteínas. O silenciamento gênico de TcasFATP foi alcançado, mas não foram observados efeitos diretos na dinâmica energética das larvas, pois os níveis de triacilglicerol e as taxas de β-oxidação não foram afetadas. Dessa forma, estudos mais detalhados com uso do silenciamento gênico serão necessários para melhor caracterização funcional das FATPs e elucidação do seu papel no metabolismo energético do inseto.

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