The appearance of adverse drug reactions is a common reason for hospitalization in Western countries. Research on underlying biological mechanisms for interindividual variability in drug response aims to better identify patients with exceptional genetic traits, disease conditions or risk of drug-drug interactions and thereby help to prevent adverse drug reactions. Active transport mechanisms are involved in the absorption and disposition of several therapeutic agents. The main objective of this thesis was to investigate factors potentially affecting transport proteins and thus contributing to variability in drug absorption and disposition. Studies of physiological, genetic, environmental, and pathological factors were included. The main focus was the two ATP-binding cassette (ABC) transporters: P-glycoprotein 170 (Pgp) and Breast Cancer Resistance Protein (BCRP). Quantification of transport protein mRNAs along the human intestine indicated that eight of the nine investigated drug transporters were expressed in a region-dependent manner. Effects of drug-drug interactions may therefore vary depending on the site of absorption. The genetic aspect was illustrated by identification of sequence variation in the gene encoding BCRP, the most highly expressed ABC transporter along the human intestine. Drug-drug interactions are important environmental causes of interindividual variability. An evaluation of the effects of Pgp-mediated drug-drug interactions showed that patients receiving Pgp inhibitors had elevated serum concentrations of the Pgp substrate digoxin and that digoxin concentrations were positively correlated with the number of co-administered Pgp inhibitors. The final topic in this thesis was that of drug-disease interactions. BCRP and Pgp were down-regulated during active inflammation in patients with ulcerative colitis. This may contribute to altered concentrations of drug in the intestinal mucosa during periods of inflammation and possibly to changes in drug absorption. To summarize, results of this thesis emphasize the complex background to the interindividual variability of drug transport proteins, where physiological, genetic, environmental and pathological factors all can contribute.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 112-125). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 5 uppsatser.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Etliche Membran Transport Proteine (MTP) sind essentiell in den Plasmodium Blutstadien, und geraten zunehmend in den Fokus der Wirkstoffentwicklung. Die physiologischen Rollen der Transporter sind jedoch oft ungeklärt. In dieser Arbeit wurden mittels experimenteller Genetik funktionelle Charakteristika der MTPs untersucht. Am Maus Parasiten Plasmodium berghei und der Plasmodium falciparum Blutstadien-Kultur wurden vier MTPs ausgewählt: ein konservierter Folat Transporter (FT2), sowie eine P. falciparum-spezifisches P-Typ ATPase und zwei essentielle MTPs (CRT und ATP4). Diese Auswahl verkörpert ein breites Spektrum an MTP Kandidaten und reflektieren zudem das Potenzial und die Grenzen funktioneller Analysen von Plasmodium MTPs mittels reverser Genetik. Für den Folat Transporter 2 (FT2) wurde eine Kombination von transgenen Strategien auf P. berghei angewandt. Durch ein endogenes tag von FT2 wurde die Lokalisierung im Apicoplast, sowie dessen Expression über fast den kompletten Zyklus hinweg gezeigt. Nach der Deletion von FT2, wiesen die Parasiten einen Defekt während der Sporulation auf. Demzufolge bilden sich nur nicht infektiöse Sporozoiten, was letztendlich zur Unterbrechung des Lebenszyklus der Parasiten führt. Eine Aminophospholipid P-Typ ATPase, wurde mittels CRISPR/Cas9 in P. falciparum genetisch deletiert und die Mutante analysiert. Im Gegensatz zu den meisten vitalen P-Typ ATPasen erweist sich das Gen in den asexuellen Blutstadien als entbehrlich. Des Weiteren bilden die MTPs ATP4 und CRT einen einflussreichen Faktor bei Malaria-Therapien. Eine umfassende Analyse von räumlichen und zeitlichen Expressionsmustern von transgenen Parasiten mit mCherry-getaggten Proteinen zeigt ein Expression der beiden MTPs über die Blutstadien hinaus, was auf zusätzliche Funktionen in den jeweiligen Stadien verweist. Diese Studie trägt, basierend auf Lokalisation, Expression und funktioneller Deletion, zur funktionellen Entschlüsselung der vier untersuchten MTPs bei. / Many membrane transport proteins (MTP) are essential for Plasmodium infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions often remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four Plasmodium MTPs. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of Plasmodium falciparum. We selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry-tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. The findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion.

Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved. One possibility is that the different subpopulations represent stages in the maturation of the endocytic pathway. Alternatively, endosome subpopulations may be specialized for different functions, such as preferential trafficking of specific endocytosed cargo. To determine whether specific receptors are targeted to distinct populations of endosomes, we have built a platform for total internal reflection fluorescence (TIRF) microscopy coupled with structured illumination capabilities named TESM (TIRF Epifluorescence Structured light Microscope.) In this study, TESM, along with standard biochemical and molecular biological tools, was used to analyze the dynamic distribution of two highly conserved Rab5 and PI3P effectors, EEA1 and Rabenosyn-5, and systematically study the trafficking of transferrin. Rabenosyn-5 is necessary for proper expression of the transferrin receptor as well as internalization and recycling of transferrin-transferrin receptor complexes. Results of combining TIRF with structured light Epifluorescence (SLE) indicate that the endogenous populations of EEA1 and Rabenoysn-5 are both distinct and partially overlapping. The application of antisense oligonucleotides as potential therapeutic agents requires effective methods for their delivery to the cytoplasm of target cells. In collaboration with RXi Pharmaceuticals we show the efficient cellular uptake of the antisense oligonucleotide sd-rxRNA® in the absence of delivery vehicle or protein carrier. In this study TIRF, SLE, and biochemical approaches were utilized to determine whether sd-rxRNA traffics and functions along specific endosomal pathways. Sd-rxRNA was found to traffic along the degradative pathway and require EEA1 to functionally silence its target. These new findings will help define the cellular pathways involved in RNA silencing. Neurotransmitter reuptake and reuse by neurotransmitter transport proteins is fundamental to transmitter homeostasis and synaptic signaling. In order to understand how trafficking regulates transporters in the brain and how this system may be disregulated in monoamine-related pathologies, the transporter internalization signals and their molecular partners must be defined. We utilized a yeast two-hybrid system to identify proteins that interact with the dopamine transporter (DAT) endocytic signal. The small, membrane associated, GTPase Rin was determined to specifically and functionally interact with the DAT endocytic signal, regulating constitutive and protein kinase C (PKC) – stimulated DAT endocytosis. The results presented in this study provide new insights into functions and components of endocytosis and enhance the understanding of endocytic organization.

Jogo patológico (JP) pode ser definido pela persistência e recorrência do comportamento de apostar em jogos de azar, apesar de prejuízos em diversas áreas da vida decorrentes dessa atividade. O JP é considerado um transtorno do controle de impulso e um modelo de dependência comportamental. Diferentes estudos têm comprovado o envolvimento de vias dopaminérgicas em dependências de substâncias e em jogadores patológicos. O transportador de doamina (DAT) é uma proteína présináptica de neurônios dopaminérgicos nigroestriatais, responsável pela recaptação da dopamina (DA) da fenda sináptica, e tem sido relatada alterações em sua densidade em dependentes de substâncias. O objetivo deste trabalho foi investigar em pacientes com jogo patológico, a densidade de DAT no estriado, através de imagens do exame de SPECT com TRODAT-1- 99mTc verificar por meio de estudo de correlação a associação entre comportamento de jogo (freqüência, tempo, dinheiro, gastos com jogo e fissura/craving) e a densidade DAT em jogadores patológicos. Foram selecionados 15 jogadores e 15 controles normais pareados para gênero, idade e escolaridade. Para inclusão ou exclusão de sujeitos foram utilizados instrumentos de verificação e principais Transtornos Psiquiátricos do Eixo 1 do DSM IV e escalas para depressão e ansiedade; para jogadores patológicos os instrumentos utilizados foram escalas para avaliação do padrão de jogo recente, para avaliação da fissura pelo jogo; para rastreamento de outras dependências comportamentais (sexo e comida). Observou-se que: 1) jogadores patológicos não apresentaram aumento de densidade DAT quando comparados a controles normais; 2) nos jogadores a densidade de DAT foi diretamente proporcional a intensidade de jogo no último mês e inversamente proporcional a auto-eficácia na abstinência do jogo. Não houve correlação significativa entre densidade de DAT e comportamentos de abuso relacionados com sexo ou comida. Desta forma, faz-se necessário estudos futuros para a avaliação da densidade de DAT no início e no fim do tratamento, a fim de verificar se a diminuição da densidade de DAT ao longo do tratamento pode ser utilizada como preditor de boa resposta e bom prognóstico em jogadores patológicos. / Pathological gambling (PG) can be defined by the persistence and recurrence of the behavior of gambling on games of chance, despite losses in many areas of life from this activity. The JP is considered a disorder of impulse control and a model of behavioral dependence. Different studies have demonstrated the involvement of dopaminergic pathways in substance dependency and pathological gamblers. The dopamine transporter (DAT) is a protein pre-synaptic dopaminergic neurons nigroestriatais responsible for the reuptake of dopamine (DA) from the synaptic cleft, and has been reported changes in the density-dependent substances. The objective of this study was to investigate in patients with pathological gambling, the density of DAT in the striatum, through examination of SPECT images with TRODAT-1 - 99mTc to verify through correlation study the association between gambling behavior (frequency, time, money spent on gambling and urge / craving) and DAT density in pathological gamblers. We selected 15 plathological gamblers and 15 controls matched for gender, age and education. For inclusion or exclusion of subjects were used verification tools and major psychiatric disorders in the DSM IV Axis 1 and scales for depression and anxiety; for pathological gamblers were the instruments used scales for assessing the standard of play recently, to evaluate the crack for the game; for tracking other behavioral addictions (sex and food). It was observed that: 1) pathological gamblers did not have increased DAT density compared with normal controls, 2) players in the density of DAT was directly proportional to the intensity of gambling in the last month and inversely proportional to self-efficacy on abstinence of gambling. There was no significant correlation between DAT density and behavior of abuse related to sex or food. Thus, it is necessary to future studies for evaluating the density of DAT at the beginning and end of treatment in order to determine whether the decrease in density during the treatment DAT can be used as predictor of good response and a good prognosis in pathological gamblers.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 6 uppsatser.