Dissertations / Theses on the topic 'SLC22A23'

According to the World Health Organization, mental disorders represent the leading cause of disability in the US generating ~58 billion dollars in medical costs annually. Additionally, among the US population, ~40 million adults suffer from an anxiety disorder and ~14 million suffer from a major depressive disorder. The association between the persistence of these neurobehavioral conditions and central nervous system (CNS) levels of biogenic amines and metabolites has been studied for half a century. Further, a number of drugs interfering with neurotransmission/metabolism are used clinically for treatment of these disorders. Recently, some members of the solute carrier (SLC) superfamily, the SLC22 transporter family, which includes organic anion transporters (Oat1, Oat3), were found to be expressed and functional on the apical membrane of the choroid plexus, a component of the blood-cerebrospinal fluid barrier. The cells of this epithelia form tight junctions, which slows penetration of solutes into the brain and limits passive efflux of endogenous solutes from the brain. Therefore, Oat1 and Oat3 are poised to play an active role in the removal of NTs and metabolites from the CSF. Thus, a better understanding of the underlying roles of OATs in regulating CNS neurotransmitters and connecting their activity to complex behaviors may result in improved understanding of the processes governing CNS homeostasis. Basal locomotor, anxiety-like and depressive-like behaviors in mice of three genotypes (WT, Oat1-/-, and Oat3-/-) across ages (3-18 mo.) were evaluated using behavioral paradigms (e.g. open field activity (OFA), light-dark (LD), marble burying (MB), and tail suspension test (TST)). Secondly, a simple high performance liquid chromatography-ultraviolet/electrochemical detection (HPLC-UV/ECD) method was developed for quantitation of monoamines and metabolites in mouse whole brain. Following completion of behavioral assessments, whole brain concentrations of monoamines and metabolites were determined using the developed method. Lastly, a novel gas chromatography tandem mass spectrometry (GC-MS/MS) method was developed for quantitation of amino acid neurotransmitters, L-glutamic acid (GA) and γ-aminobutyric acid (GABA), in mouse whole brain. The developed method was used for measurement of whole brain concentrations of GA and GABA in a small subset of WT, Oat1-/-, and Oat3-/- mice at 3 and 18 mo.

The organic cation transporters (OCTs) play a critical role in the absorption, distribution and elimination of many drugs, hormones, herbal medicines, and environmental toxins. Given the broad substrate specificity of OCTs, they fall victim to the high susceptibility for contributing to harmful drug-drug interactions. Further defining how human (h)OCTs mechanistically bind to its broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCTs. The goal of the current study was to elucidate the critical amino acid residues for transporter-substrate binding interactions on human (h)OCT1 and 2 utilizing in silico molecular modeling techniques (homology modeling and automated docking), as well as in vitro mutagenesis and kinetic transport experiments. Three-dimensional homology models were generated for hOCT1 and 2 using Piriformospora indica phosphate transporter (PiPT) serving as template. A putative binding pocket was identified and used to dock the prototypical substrate MPP+. Docking studies revealed five residues for each transporter (hOCT1 and hOCT2) that may be critical for substrate-transporter interactions. The in silico data was used to guide subsequent in vitro site-directed mutagenesis and kinetic analysis. Four hOCT1 mutants (Gln241Lys, Thr245Lys, Tyr361Ala, and Glu447Lys) and three hOCT2 mutants (Gln242Lys, Tyr362Phe, and Tyr362Ala) showed complete loss of MPP+ transporter activity. Decreased affinity for MPP+ was observed for Phe244Ser and Thr245Ser in hOCT1, and Tyr245Ala in hOCT2. All amino acid residues highlighted in the in vitro experiments may be potentially critical for substrate-transporter interactions particularly Tyr361, Phe244 and Thr245 in hOCT1; and Tyr362 and Tyr245 in hOCT2. Docking of known structurally divergent hOCT1 and hOCT2 substrates revealed similar binding interactions as that identified for MPP+, albeit with some unique residues, suggesting the presence of a large central cavity within both transporters. Through the combination of in silico and in vitro experiments, a putative binding pocket was defined and several residues important for substrate-transporter interaction were identified and verified for hOCT1 and hOCT2. Further defining how OCTs biochemically interact with their broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCT1 and OCT2.

Actualmente la diabetes mellitus tipo 2 es un problema de salud pública mundial, debido a factores como el estilo de vida de la población, con tendencia al sedentarismo y consumo de comidas procesadas, lo que contribuye a su actual incremento. El tratamiento de primera línea es la administración de metformina, un fármaco antihiperglucémico cuya farmacodinámica depende de transportadores como los OCTs, tanto para su ingreso a las células como para su excreción vía urinaria. La gran variabilidad interindividual de la concentración plasmática de metformina indica una variación en la tasa de excreción del fármaco, reportado por distintos autores en otras poblaciones, la cual es causada por variaciones en genes encargados de su excreción, como el gen SLC22A2, codificante del trasportador catiónico OCT2. En el presente estudio se estandarizó una técnica para la unión de productos de PCR diferentes, haciendo uso de la técnica OE-PCR, con la cual se unieron los exones 2, 3 y 4 del gen SLC22A2. Se caracterizó el exón 4 del gen SLC22A2 en peruanos nativos y mestizos, detectándose en un 6.25% de individuos el polimorfismo 808C>A en heterocigosis (uno de la población nativa y dos de la mestiza), el 93.75% de individuos restantes presentaron el genotipo 808AA, estos resultados concuerdan con los que se encuentran en la base de datos 1000 genomas. Esta variante tiene una gran implicancia farmacológica y ha sido reportada en diversos estudios como causante de un menor aclaramiento del fármaco, conllevando a un aumento de su concentración plasmática, pudiendo ocasionar efectos adversos en pacientes. Este estudio sirve como base para análisis más amplios de este y otros genes en la población peruana con implicancia farmacológica para enfermedades como la diabetes tipo 2.
Tesis

SUMMARYThe diversity of cancer molecular origins associated with the genetic variability of patients has encouraged the development of chemotherapeutic treatments adapted not only to the target tumor, but also to a specific patient. This personalized strategy is based on cancer biomarkers allowing a better identification and characterization of each tumor where predictive biomarkers provide the distinction between various factors indicative of the response to the treatment. In this context, several studies highlighted the role of the solute carrier transporter family 22 (solute carriers 22 or SLC22) in the uptake of platinum anticancer drugs. This mechanism being not well understood, our work intends to establish the potential role of SLC22 member A18 (SLC22A18) as predictive biomarker in the aim to help to a better targeted chemotherapeutic strategy for each patient. We optimized a system overexpressing SLC22A18 stably or transiently in HeLa cancer cell line. SLC22A18 expression was confirmed by qRT-PCR, western blotting, microscopy and flow cytometry. The cell lines were treated with taxane, anthracyclin, vinca alkaloid and nitrosoureas anticancer drug families. We showed that doxorubicin, camptothecin, chloroquine, tetracycline and carmustin had no effect on the cell viability assays suggesting that they are not substrates of SLC22A18. Interestingly, the cell line was sensitized in the presence of antimitotic drug with a sensitivity factor of 2.7 in the presence of paclitaxel, 1.4 with docetaxel, 1.8 with vinblastin and 2.2 in the presence of vincristine. To confirm these results, we elaborated a SLC22A18 knockdown cell line in HS683 cells using siRNA technology. The downexpression of SLC22A18 was correlated to a tendency to resist to the accumulation of paclitaxel thereby confirming the previous results. Simultaneously, a knockout cell line was established using the transcription activator-like effectors nuclease (TALEN) technology in U373 cell line. Our studies constitute a robust base of knowledge for further investigation on SLC22A18 transporter as a predictive biomarker promoting antimitotic treatment in tumors where this transporter is detected.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

A leucemia mieloide crônica (LMC) é uma expansão clonal da célula tronco hematopoética, traduzindo-se por hiperplasia mieloide, leucocitose, neutrofilia, basofilia e esplenomegalia. O cromossomo Filadélfia é característico da doença, sendo produto da translocação t(9:22)(q34;q11), resultando na fusão dos genes ABL e BCR. Esta fusão gera um gene híbrido que codifica uma proteína com elevada atividade tirosinoquinase e tem um papel central na patogenia da LMC. O mesilato de imatinibe (MI) é um derivado da fenilaminopirimidina que inibe a proteína tirosinoquinase BCR-ABL1 in vitro e in vivo. O MI interage com transportadores de membrana de influxo, como o organic carion solute carrier 22 ,member 1 (SLC22A1,hOCT1); e de efluxo, como ATP binding cassette B1 (ABCB1, MDR1, P-gp). Os polimorfismos ABCB1 c.1236C>T, C.3435C>T e c.2677G>T/A têm sido associados com a alteração da função da P-gp. Este estudo teve por objetivo investigar a relação da expressão do RNAm de ABCB1 e SLC22A1 com marcadores de resposta ao tratamento com MI e avaliar a atividade funcional da P-gp em células mononucleares de pacientes com diferentes haplótipos para os polimorfismos ABCB1 c.1236C>T, c.3435C>T e c.2677G>T/A. Foram incluídos 118 pacientes com LMC para o estudo da expressão do RNAm de SLC22A1 e ABCB1 e para o estudo da atividade da P-gp foram selecionados 28 pacientes de acordo com os haplótipos dos polimorfismos ABCB1 c.1236C>T, c.3435C>T e c.2677G>T/A. Para o estudo da expressão do RNAm de SLC22A1 e ABCB1 foram constituídos dois grupos: Grupo 1 com 70 pacientes com resposta citogenética completa com a dose padrão de MI (400 mg/dia de MI) em até 18 meses e, Grupo 2 com 48 pacientes sem resposta citogenética completa com a dose inicial de 400 mg/dia de MI ou que perderam esta resposta ao longo do tratamento. Para o estudo da atividade funcional da P-gp, dos 118 pacientes incluídos, foram selecionados 10 pacientes que apresentaram o haplótipo 1236CC/3435CC/2677GG, 10 pacientes que apresentaram o haplótipo 1236CT/3435CT/2677GT e 8 pacientes que apresentaram o haplótipo 1236TT/3435TT/2677TT. A resposta ao tratamento foi avaliada segundo os critérios da European LeukemiaNet. Amostras de sangue foram obtidas para: quantificação de BCR-ABL1, extração do RNAm total, análise citogenética de banda G, dosagem da concentração plasmática de MI e análise da atividade e expressão da P-gp. A análise da expressão dos genes ABCB1 e SLC22A1 foi feita por PCR em tempo real, a análise da atividade e expressão da P-gp foram feitas por citometria de fluxo e a dosagem da concentração plasmática de MI foi realizada por eletroforese capilar. Resultados: A expressão de ABCB1 e SLC22A1 foi analisada nos 118 pacientes incluídos e foi similar entre os grupos de resposta. A elevada expressão do gene SLC22A1 foi associada àqueles pacientes que alcançaram a resposta molecular maior (RMM) no grupo respondedor (P=0,009). Não houve associação entre a expressão de ABCB1 e a resposta ao MI. Nenhum dos genes foi associado à resposta molecular completa (RMC). No estudo da atividade da P-gp foi observada uma maior atividade nos pacientes que apresentavam o haplótipo 1236CC/3435CC/2677GG quando comparado àqueles que possuíam o haplótipo com alelo mutado. Não houve diferença na expressão do RNAm dos genes SLC22A1 e ABCB1, expressão da P-gp e concentração plasmástica de MI entre os grupos de haplótipos. Os pacientes que não alcançaram a RMM apresentaram uma maior taxa de efluxo mediado pela P-gp quando comparado aos indivíduos que alcançaram esta resposta (64,7% vs. 45,7%; P=0,001). Os indivíduos que alcançaram a RMM e RMC apresentaram maior mediana de expressão do gene SLC22A1. Os pacientes sem RMM apresentaram menor concentração plasmática de MI quando comparados aos que alcançaram esta resposta (0,51 µg/mL vs. 1,42 µg/mL; P=0,001). Não foi observada associação entre a concentração plasmática de MI e a RMC. Em conclusão os pacientes respondedores a dose padrão de 400mg/dia de MI e que alcançaram a RMM apresentam maior expressão de RNAm de SLC22A1 e os portadores dos haplótipos 1236CT/3435CT/2677GT e 1236TT/3435TT/2677TT exibem menor efluxo mediado pela P-gp apresentando maior frequência de RMM.
Chronic myeloid leukemia (CML) is a clonal expansion of hematopoietic stem cell, translating into myeloid hyperplasia, leukocytosis, neutrophilia, basophilia and splenomegaly. The Philadelphia chromosome is characteristic of the disease, being the product of the translocation t(9:22)( q34,q11), resulting in the fusion of the BCR and ABL genes. This fusion generates a hybrid gene that encodes a protein with elevated tyrosine kinase activity and plays a central role in the pathogenesis of CML. Imatinib mesylate (IM) is a derivative of fenilaminopirimidine that inhibits BCR-ABL1 fusion protein tyrosine kinase in vitro and in vivo. IM interacts with uptake membrane transporters, such as cation organic solute carrier 22, member 1 (SLC22A1, hOCT1) and efflux as ATP binding cassette B1 (ABCB1, MDR1,P-gp). ABCB1 polymorphisms c.1236C>T,c.3435C>T and c.2677G>T/A have been associated with altered function of P-gp. This study aimed to investigate the relationship between mRNA expression of ABCB1 and SLC22A1 with markers of response to treatment with IM and evaluate the functional activity of P-gp in mononuclear cells of patients with different haplotypes for ABCB1 c.1236C>T, c.3435C>T and c.2677G>T/A polymorphisms. This study included 118 patients with CML to study the mRNA expression of SLC22A1 and ABCB1 and to study the P-gp activity, 28 patients were selected according to the haplotypes of ABCB1 c.1236C>T, c.3435C>T and c.2677G>T/A polymorphisms. To study the mRNA expression of SLC22A1 and ABCB1, two groups were constituted: Group 1 with 70 patients with a complete cytogenetic response with standard-dose IM (400 mg/day) in 18 months, and group 2 with 48 patients without complete cytogenetic response with the initial dose of IM (400 mg/day) or have lost this response during treatment. To study the P-gp functional activity, 10 patients with haplotype 1236CC/3435CC/2677GG, 10 patients with haplotype 1236CT/3435CT/2677GT and 8 patients with haplotype 1236TT/3435TT/2677TT were enrolled. Treatment response was assessed according to European LeukemiaNet criteria. Blood samples were obtained for: quantification of BCR-ABL1, mRNA extraction, G band cytogenetic analysis, measurement of IM plasma levels and P-gp activity and expression. The ABCB1 and SLC22A1 gene expression analysis was made by real-time PCR, analysis of P-gp activity and protein expression were performed by flow cytometry and determination of plasma Levels of IM was performed by capillary electrophoresis. Results: Expression of ABCB1 and SLC22A1 were analyzed in 118 patients included and was similar between the response groups. Higher expression of the SLC22A1 gene was associated with those patients who achieved a major molecular response (MMR) in the responder group (P=0.009). There was no association between ABCB1 expression and IM response. None of the studied genes was associated with complete molecular response (CMR). In the study of P-gp activity we observed greater activity mediated by P-gp in patients with 1236CC/3435CC/2677GG haplotype when compared to those with the mutated allele. There was no difference in mRNA expression of SLC22A1 and ABCB1 genes, P-gp expression and IM plasma levels between haplotypes groups. Patients who did not achieve MMR showed a higher rate of efflux mediated by P-gp compared to individuals who did achieve this response (64.7% vs. 45.7%, P=0.001). Individuals who achieved MMR and CMR had higher median of SLC22A1 expression. Patients without MMR had lower IM plasma levels compared with those who achieved this response (0.51 µg/mL vs. 1.42 µg/mL, P=0.001). No association was observed between IM plasma levels and CMR. In conclusion patients responders to standard dose of IM (400 mg/day) and who achieved MMR have higher SLC22A1 mRNA expression and the carriers of 1236CT/3435CT/2677GT 1236TT/3435TT/2677TT haplotypes exhibit lower efflux mediated by P-gp with higher frequency of MMR.

Les inhibiteurs de tyrosine kinases (ITKs) sont une nouvelle classe thérapeutique ayant connu un grand essor ces dix dernières années. Inhibiteurs compétitifs de l’adénosine triphosphate (ATP), ils sont utilisés dans le traitement de nombreux cancers dans lesquels une dérégulation de tyrosine kinases a été mise en évidence. Malgré une efficacité prouvée, des cas de résistance sont rapportés, en particulier avec l’exemple de la leucémie myéloïde chronique (LMC) et le traitement par ITK. Cette variabilité inter-individuelle peut être due à des mécanismes de résistance propre de la cellule tumorale ou à des variations dans les paramètres pharmacocinétiques de la molécule. De nombreuses études ont analysé l’impact de polymorphismes (SNPs) dans des gènes codants pour les déterminants pharmacocinétiques et pharmacodynamiques des ITKs. Nous avons analysé l’impact de SNPs sur l’obtention de la réponse moléculaire majeure à 1 an dans 2 cohortes de patients atteints de LMC et traités par imatinib. C1236T, G2677T/A et C3435T, 3 SNPs du gène MDR-1 codant pour la glycoprotéine P et les SNPs de la région codante du gène SLC22A1 à l’origine du transporteur d’influx hOCT1. L’impact bénéfique de l’allèle 1236T ou haplotype *4 et l’impact péjoratif de l’allèle 2677G ou haplotype *1, retrouvés dans la 1ère cohorte n’ont pas été retrouvés dans la 2ième cohorte suggérant un impact mineur voire nul de ces derniers sur la réponse à l’imatinib. L’impact des SNPs de SLC22A1 observés dans la 2ième cohorte nécessite d’être confirmé. Des travaux supplémentaires à plus grande échelle, selon des critères nécessitant d’être harmonisés, sont nécessaires avant d’espérer pouvoir aboutir à une «médecine personnalisée» pour l’imatinib mais également de façon générale pour l’ensemble des ITKs
Tyrosine kinases inhibitors (TKIs) are a new class of drugs having bloomed over the past decade. As competitive inhibitors of the adenosine triphosphate, they are used in the treatment of many cancers in which deregulation of tyrosine kinases has been demonstrated. In spite of dramatic efficacy, cases of resistance have been reported particularly with chronic myeloid leukemia (CML) and TKI treatment. This inter-individual variability may be due to mechanisms of intrinsic resistance of tumor cells or changes in the pharmacokinetic parameters of the molecule. Numerous studies have analyzed the impact of polymorphisms (SNPs) in genes coding for pharmacokinetic and pharmacodynamic determinants. We analyzed the impact of SNPs on major molecular response at 1 year in 2 cohorts of patients with CML treated with imatinib. C1236T, G2677T/A, C3435T, three SNPs in the MDR-1 gene encoding P-glycoprotein and SNPs in the coding region of the SLC22A1 gene encoding hOCT1. The protective impact of the 1236T allele or haplotype*4 and the pejorative impact of the 2677G allele or haplotype*1, found in the 1st cohort, were not replicated in the 2nd cohort, suggesting minor or no impact on the response to imatinib. The impact of SLC22A1 SNPs observed in the 2nd cohort needs to be confirmed. Further works on a larger cohort, according to criteria that need to be harmonized, are necessary before we reach a “personalized medicine” for imatinib but also for all TKIs

INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas.
BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.

Inflammatory bowel disease (IBD) is a chronic disease which steadily increases worldwide with the highest prevalence in Canada. Genetic susceptibility is considered to be an important factor in causing IBD. Organic cation transporters, SLC22A4 and SLC22A5 have been associated to IBD multiple times. Recently, SLC22A23, a novel gene that encodes for an organic cation membrane transporter protein has also been associated to IBD however; neither its gene structure nor its functions has been characterized. The aim of this study was to characterize the genomic structure of SLC22A23 gene using bioinformatics analysis, determine the tissue expression, characterize the location of the protein and perform functional studies using Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry. We have identified the chromosomal location, the gene neighborhood and the genomic structure of human SLC22A23.The result of this study indicates that SLC22A23 gene is a membrane transporter and it is abundantly expressed in the intestine.
October 2015

Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

博士
高雄醫學大學
醫學研究所
99
Objective: To investigate variants of MAOA, SLC2A9 and SLC22A12 associated with higher hyperuricemic risk to develop gout/chronic tophaceous gout. Design: Case-control study. Setting: Hospital- and population -based study from Taiwan and Solomon Islands. Participants: Gout patients (n=374, 157 and 69) and non-gout controls (n=604, 401 and 406) for Taiwan aborigines, Taiwan Han Chinese and Solomon Islanders were recruited. Main outcome measure: MAOA, SLC2A9 and SLC22A12 gene variants were genotyped. Results: In the first study, a synonymous MAOA rs1137070 C allele was associated with the risk of having gout specifying in aborigines (odds ratio [OR] 1.46, 95% confidence interval [CI] 1.11–1.91, P = 4.0× 10-5). MAOA enzyme activity by rs1137070 alleles showed graded associations with hyperuricemia and gout (P for trend=1.53 × 10-6 versus wild-type T allele). Secondly, patients with SLC2A9 rs3733591 Arg265His risk C-allele consistently in both Taiwan Han Chinese and Solomon Islander populations had higher risk for tophaceous gout (OR 2.05, 95% CI 1.11-3.77, P=0.0044; OR 2.08, 95% CI 1.02-4.23, P=0.0184). Thirdly, gout risk was higher concordantly for patients harbouring a common C-allele of SLC22A12 rs475688 in both studied groups (OR 1.98, 95 % CI 1.36-2.88, P=0.0054; OR 1.77, 95% CI 1.10-2.84, P=0.0115, respectively). Moreover, being homozygote at two-locus SLC2A9 rs3733591/ SLC22A12 rs475688 CC/CC-risk score was maximally 14.12-fold in Taiwan Han Chinese (95% CI, 3.57-55.89, P=0.0002) and was 6.98-fold in Solomon Islanders (95% CI 1.02-47.58, P=0.0472) gout risk. Importantly, the interaction term modeled between rs3733591 and rs475688 was significant association with gout in Han (P for interaction=0.0295) but not in Solomon Islanders (P for interaction=0.8622). Conclusion: MAOA gene variants with innate and adaptive immunity may contribute to the development gout in Taiwan aborigines. SLC22A12 and SLC2A9 gene variants were associated with higher hyperuricemic risk to develop gout and to progress tophaceous gout in Han Chinese and replicated in Solomon Islanders.

Siu Shu Shun.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 100-106).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Contents --- p.ii
Abstract / 摘要 --- p.iv
Abbreviations --- p.vi
List of figures --- p.vii
List of tables --- p.x
Chapter Chapter 1: --- Introduction
Chapter 1.1 --- "Human EST sequencing project, the role and goal" --- p.1
Chapter 1.2 --- Human liver cDNA sequencing --- p.2
Chapter 1.3 --- The role of membrane-associated proteins in hepatocellular functions --- p.3
Chapter 1.3.1 --- Outline of the liver function --- p.3
Chapter 1.3.2 --- Basic structure of hepatocyte --- p.4
Chapter 1.3.3 --- Category of membrane associated proteins --- p.5
Chapter 1.4 --- Identification of human OAT2 gene --- p.7
Chapter 1.5 --- The multispecific transporter family --- p.8
Chapter 1.5.1 --- Classification --- p.8
Chapter 1.5.2 --- The human OAT family --- p.9
Chapter 1.6 --- The characteristics of rat multispecific OAT2 --- p.11
Chapter 1.7 --- Clinical significance of organic anion transport proteins --- p.14
Chapter Chapter 2: --- Materials and Methods
Chapter 2.1 --- Human liver EST sequencing project --- p.16
Chapter 2.1.1 --- Plating out the adult human liver phage library --- p.16
Chapter 2.1.2 --- PCR detection and amplification of the cDNA clone --- p.17
Chapter 2.1.3 --- Automatic cDNA sequencing --- p.18
Chapter 2.2 --- Cloning of hOAT2 gene into TA cloning vector pT-Adv --- p.19
Chapter 2.2.1 --- Amplification of hOAT2 by PCR --- p.19
Chapter 2.2.2 --- Ligation reaction --- p.19
Chapter 2.2.3 --- Transformation of recombinant plasmid into competent cells --- p.20
Chapter 2.3 --- Sequence analysis and structural prediction --- p.20
Chapter 2.4 --- Cloning of the hOAT2 gene into the pQE30 expression vector --- p.21
Chapter 2.4.1 --- PCR amplification and restriction endonuclease cutting --- p.21
Chapter 2.4.2 --- Gene clean --- p.22
Chapter 2.4.3 --- Preparation of bacterial competent cells --- p.23
Chapter 2.5 --- Small scale synthesis of plasmid DNA --- p.24
Chapter 2.6 --- Large scale synthesis of plasmid DNA --- p.25
Chapter 2.7 --- Cloning of the hOAT2 gene into the pSecTag2B mammalian expression vector --- p.26
Chapter 2.8 --- Cloning of the hOAT2 gene into the pEGFP-C2 fluorescent vector --- p.27
Chapter 2.8.1 --- Tissue culture and transfection --- p.27
Chapter 2.8.2 --- Fluorescence microscopy examination --- p.28
Chapter 2.9 --- Chromosomal mapping of the hOAT2 gene --- p.29
Chapter 2.9.1 --- Somatic cell hybrids mapping --- p.29
Chapter 2.9.2 --- Radiation hybrids mapping --- p.29
Chapter 2.10 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.30
Chapter 2.11 --- Western hybridization --- p.32
Chapter 2.11.1 --- Preparation of anti-hOAT2 antibodies --- p.32
Chapter 2.11.1.1 --- Synthetic peptide conjugation --- p.32
Chapter 2.11.1.2 --- Immunizing rabbit polyclonal antibodies for human OAT2 --- p.32
Chapter 2.11.1.3 --- Purification of the rabbit polyclonal IgG antibodies --- p.33
Chapter 2.11.2 --- Western blot analysis --- p.33
Chapter 2.11.2.1 --- Protein isolation from rat liver --- p.33
Chapter 2.11.2.2 --- Prote in preparation from cell lysate --- p.34
Chapter 2.11.2.3 --- Quantitation of total proteins by Bradford protein assay --- p.35
Chapter 2.11.2.4 --- Blotting and hybridization --- p.35
Chapter Chapter 3: --- Results
Chapter 3.1 --- Catalogue of the 500 liver ESTs --- p.37
Chapter 3.2 --- Nomenclature of human NLT gene --- p.47
Chapter 3.3 --- Cloning and characterization of the hOAT2 sequence --- p.48
Chapter 3.3.1 --- Isolation of hOAT2 cDNA from human liver cDNA library --- p.48
Chapter 3.3.2 --- The primary and secondary structural analysis of hOAT2 --- p.53
Chapter 3.3.3 --- Motif search and prediction --- p.61
Chapter 3.3.4 --- Homology alignment --- p.64
Chapter 3.4 --- Chromosomal mapping of hOAT2 gene --- p.67
Chapter 3.4.1 --- Somatic cell hybrid mapping of hOA T2 gene --- p.67
Chapter 3.4.2 --- Radiation hybrid mapping of hOA T2 gene --- p.69
Chapter 3.4.3 --- Identification of partial human genomic sequence --- p.73
Chapter 3.5 --- Detection of the hOAT2 gene expression in human tissues by RT- PCR assay --- p.76
Chapter 3.6 --- Detection of subcellular localization of hOAT2 protein by conjugating fluorescence protein --- p.81
Chapter 3.7 --- Immunodetection of protein extracts from cultured cells --- p.83
Chapter Chapter 4: --- Discussion
Chapter 4.1 --- Characterization of the hepatocellular ESTs --- p.85
Chapter 4.1.1 --- Classification and frequency distribution of the 500 ESTs --- p.85
Chapter 4.1.2 --- The expression pattern of membrane associated proteins --- p.87
Chapter 4.2 --- Tissue distribution and expression profiles of hOAT2 --- p.88
Chapter 4.3 --- HOAT2 in fetal development --- p.89
Chapter 4.4 --- Predicting the topology of membrane proteins --- p.90
Chapter 4.5 --- Chromosomal mapping of human OAT2 --- p.91
Chapter 4.6 --- Possible functions of hOAT2 --- p.93
Chapter 4.6.1 --- Hepato-renal relation --- p.93
Chapter 4.6.2 --- Substrate diversity --- p.95
Chapter 4.7 --- Fluorescence detection for subcellular localization --- p.96
Chapter 4.8 --- Conclusion --- p.97
Chapter 4.9 --- Further aspects --- p.99
References --- p.100
Appendix --- p.107

碩士
國立臺灣大學
分子醫學研究所
97
Mutations in the SLC22A5 gene, which encodes the plasma membrane carnitine transporter OCTN2, cause primary carnitine deficiency (PCD). Currently, PCD is screened in newborns using free carnitine level as a marker. However, owing to the high free carnitine level in the maternal circulation, the affected babies may not have a free carnitine level low enough to be picked up by screening. Previously, the OCTN2 gene c.981 C>T (p.R254X) mutation was found to be common in the Southern Chinese population. Therefore, in this study we want to see if molecular diagnosis could enhance newborn screening of PCD. In this study, we analyzed blood spot DNA OCTN2 gene p.R254X mutation. We first analyzed 348 random anonymous control DNA samples to see the prevance of this mutation in the population. We then analyzed 48 blood spot samples in which free carnitine level was lower than 11µM (the standard cut off for free carnitine was lower than 6.44 μΜ). We found that among the three methods for blood spot DNA extreaction (the methanol method, boiling method, and QIAamp method); the QIAmp method gave the best result. We then found two p.R254X heterozygotes in the 348 control DNA (1in 174); another two p.R254X heterozygotes in the 48 samples with low free carnitine level (1 in 24). In comparison to previous data, newborns with low free carnitine level did have a higher prevalence of OCTN2 gene p.R254X mutation (p=0.01996). Babies who had both low free carnitine and OCTN2 mutation should have a higher chance to be a patient of primary carnitine deficiency, and their disease status should be determined. This method, therefore, increases the sensitivity of detecton of PCD without increase the false positive rate of the screening.

碩士
臺北醫學大學
臨床醫學研究所
97
Serum uric acid is the degradation product of purines (ATP, GTP & nucleic acid). Serum uric acid level is maintained by urate synthesis and excretion. Whenever hyperurecemia happened, the joint inflammatory change, cause a lot of pain. The risk of deposition of uric acid around joints will increase, thus cause joint, tendon destruction and disability. The tophus formation and joint destructive process induce very severe pain and disability, which is known as gouty arthritis. For a long time, uric acid metabolitic process is not been fully understood. Is it really the only final excretional product or if it still has some possible usefulness? The answer is quite clear, under some certain circumstances, it can be function similar to vitamin C, as a potent antioxidant. Also, urate can maintain blood pressure under low salt conditions via stimulation of the reninangiotensin system through a mechanism that is still poorly understood. The renal tubule apical protein, URAT1 (coded by SLC22CA12) was recently proposed to be the major absorptive urate transporter protein in the kidney regulating blood urate levels. A study of the Janpanese genetic variations in SLC22A12 gene, rs893006 polymorphism (GG, GT and TT) in a total of 326 Japanese subjects was published. The significant correlation between single nucleotide polymorphism (SNP) in the urate transporter gene SLC22CA12 was found to be associated with the elevated serum uric acid levels. In Taiwanese aborigines, has a remarkably high prevalence of hyperuricemia and gout. We collected 368 volunteers blood samples, which including 175 cases of male and 193 cases of female. (Ataya, Bunun, Paiwan, and general Taiwanese)(including 133 cases of general population of taiwanese as control group, 235 cases are the Taiwanese aborigines(Atayal),) The genomic DNA from peripheral blood lymphocytes will be collected, and use for genotyping of the rs893006 polymorphism in SLC22A12 gene, comparing the difference between Taiwanese aborigines and the general population. Otherwise, we will also compare the difference between the plasma level of creatinine, fasting plasma glucose level, triglyceride, serum cholesterol, BMI and the serum uric acid level.