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 (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 106-116.

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.

Made available in DSpace on 2014-12-17T15:32:21Z (GMT). No. of bitstreams: 1 ClarissaFD_DISSERT.pdf: 1047575 bytes, checksum: 1162ac27915144b24bb631aefd3e421b (MD5) Previous issue date: 2012-02-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The Epidermoid Carcinoma (EC) is the most common lesions located in the region of the head and neck and, despite advances in treatment modalities, the prognosis is still poor. The malignant cells show an increase in glucose uptake, process mediated by glucose transporters (GLUTs). Increased expression of GLUT 1 and GLUT 3 is related to the aggressive behavior of this lesion. The aim of this study was to evaluate, through immunohistochemistry, the expression of GLUTs 1 and 3 in EC of the lower lip. The sample consisted of 40 cases of EC of the lower lip, of which 20 had regional lymph node metastasis and the remaining 20 with absence of metastasis. The percentages of immunostained cells in front of tumor invasion and in the center of tumor were evaluated. These results were related to the presence and absence of lymph node metastasis, TNM classification and histological grading. The percentage of cytoplasmic/membranous expression of GLUT 1 ranged from 77.35% to 100%, while for GLUT 3 this value ranged from 0.79% to 100%. As for nuclear staining for GLUT 1, this percentage ranged from 0 to 0.42%, however. GLUT 3 showed only one case with nuclear staining. Despite the significant expression of tumor cells related to the proteins studied, we observed no statistically significant relationship between the variables and the antibodies analyzed, regardless of the region evaluated. However, there was a moderate positive correlation between cytoplasmic/membranous immunoexpressions of GLUT 1 in invasion front and in the tumor center (r = 0.679, p <0.001). Similarly, moderate positive correlation was found between the nuclear immunoexpressions of GLUT 1 in the invasion front and in the tumor center (r = 0.547, p <0.001). For GLUT 3, was also observed a moderate statistically significant positive correlation between cytoplasmic/membranous expression in tumor invasion front and in tumor center (r = 0.589, p <0.001). We also observed that the immunoreactivity for GLUT 1 was higher than GLUT 3 expression in invasion front (p <0.001) and tumor center (p <0.001). From these results, this study suggests that tumor hypoxia is a remarkable characteristic of the EC of the lower lip and GLUT 1 may be primarily responsible for glucose uptake into the interior of the malignant cells / O Carcinoma epiderm?ide (CE) est? entre as les?es mais comuns localizadas na regi?o de cabe?a e pesco?o e apesar dos avan?os nas modalidades de tratamento, o progn?stico ainda ? pobre. As c?lulas malignas apresentam um aumento na absor??o de glicose, processo esse mediado pelos transportadores de glicose (GLUTs). O aumento da express?o de GLUT 1 e GLUT 3 encontra-se relacionado com o comportamento agressivo dessa les?o. O objetivo desse estudo consistiu em avaliar, atrav?s de estudo imuno-histoqu?mico, a express?o dos GLUTs 1 e 3 em CE de l?bio inferior. A amostra foi composta por 40 casos de CE de l?bio inferior, dos quais 20 apresentavam met?stase linfonodal regional e os 20 restantes com aus?ncia de met?stase. Foram avaliados os percentuais de c?lulas imunomarcadas no front de invas?o e no centro do tumor, esses resultados foram relacionados com a presen?a e aus?ncia de met?stase linfonodal, estadiamento cl?nico TNM e grada??o histol?gica. O percentual de marca??o citoplasm?tica/membranar para GLUT 1 variou de 77,35% a 100%, j? para GLUT 3 esse valor variou de 0,79% a 100%. Quanto ? marca??o nuclear para GLUT 1, este percentual variou de 0 a 0,42%, no entanto, GLUT 3 apresentou apenas um caso com marca??o nuclear. Apesar da expressiva marca??o das c?lulas tumorais frente aos marcadores estudados, n?o foi poss?vel observar rela??o estatisticamente significativa entre as vari?veis estudadas e os marcadores analisados, independente da regi?o avaliada. Contudo, foi observada moderada correla??o positiva estatisticamente significativa entre as imunoexpress?es citoplasm?tica/membranar de GLUT 1 no front de invas?o e no centro do tumor (r=0,679; p<0,001). De forma similar, foi constatada moderada correla??o positiva entre as imunoexpress?es nucleares de GLUT 1 no front de invas?o e no centro tumoral(r=0,547; p<0,001). Para GLUT 3, tamb?m foi observada moderada correla??o positiva entre as imunoexpress?es membranar/ citoplasm?tica dessa prote?na no front de invas?o e no centro tumoral (r=0,589; p<0,001). Foi observado, ainda, que a imunoexpress?o de GLUT 1 em rela??o a GLUT 3 foi maior tanto no front de invas?o (p < 0,001) como no centro do tumor (p<0,001). A partir desses resultados, conclui-se que a hip?xia tumoral ? uma caracter?stica marcante no CE de l?bio inferior e GLUT 1 pode ser o principal respons?vel pela capta??o de glicose para o interior das c?lulas malignas

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.