The norepinephrine transporter (NET) clears norepinephrine (NE) from the synapse after vesicular release. NET is a target of the psychostimulant amphetamine (AMPH). We have recently shown that AMPH alters trafficking of the transporter, causing a net decrease in surface NET in the monoaminergic CAD cell line. In this study we demonstrate, by confocal imaging of immunostained superior cervical ganglion (SCG) neurons, that AMPH causes an increase in NET levels inside terminal boutons. Further, the intracellular compartment in which NET accumulates upon AMPH exposure is not known; such information would inform investigation of the mechanism by which the drug affects NETs cellular distribution. We show that after AMPH treatment, NET co-fractionates with both Rab4 and Rab11, and that AMPH increases co-localization of NET with these endosomal markers as indicated by the intensity correlation quotient (ICQ). Finally, we show that the functions of both GTPases are involved in AMPH-regulated NET trafficking by transfection of dominant negative (DN) constructs followed by cell-surface biotinylation. Our results support the conclusion that AMPH-regulated NET trafficking occurs through endosomes.

Cancer remains the most cause death disease in Taiwan at least ten years. Liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), is the most common cause of cancer mortality in men and the second most in women. Not only liver section and liver transplantation are used in HCC therapy but also local ablation therapy and transarterial therapy. Transarterial chemoembolization (TACE) is one of the local therapies that inject chemotherapeutic drugs directly into liver tumor. However, drug resistance is the mainly restriction in patient after chemotherapy. Moreover, it is known that drug resistance was associated to over-expression of certain ABC transporter genes, especially ABCB1, ABCG2, and ABCC family in cancer cell and those ABC transporters were also expressed in liver. Base on clinical study, they use 5-fluororuacil, cisplatin and mitomycin-C for liver cancer treatment. In this study, we hypothesized that cancer therapies may be augmented through blocked the drug efflux ABC channels with the ABC transporter inhibitors such as verapamil. The associations among drug treatments, inhibitor incorporation and the expression of ABC transporters were evaluated in HepG2 and Hep3B cells. MTT assay demonstrated that the cell viability was considerable decreased by treating triple drugs with verapamil. RT-PCR data showed that ABC transporters mRNA expression has no significantly change. However, membrane ABCB1 and ABCG2 were induced after drugs and inhibitors treatment either 1 or 24 hours by flow cytometry analysis. P-glycoprotein functional assay also showed p-glycoprotein was inhibited by verapamil, and hence Rhodamine 123 retention was increased. Taken together, there are different response of ABC transporters in HepG2 and Hep3B after drugs and inhibitors treatment. Membrane ABCB1 and ABCG2 were induced by drugs and inhibitors treatment. However, p-glycoprotein¡¦s function was restrained simultaneously by inhibitors treatment. Therefore, verapamil can enhance cell death by inhibiting ABC transporters and its cytotoxic effect rather than the increased expression of ABC transporters. This finding might provide a better way in liver cancer therapy.

I denna uppsats ställs  frågan hur Kristianstads infrastruktur motsvarar den fysiska utformningen som förekommer i framgångsrika cykelstäder, där andelen cykelpendlare är hög. Studien går därför ut på att undersöka vad begreppet cykelvänlig stad innebär och att analysera Kristianstads cykelnät, vilket gör det möjligt att föra en diskussion om hur Kristianstad skulle kunna förbättra de fysiska förutsättningarna för ökad cykelpendling. Examensarbetet har bedrivits inom ramen av en fallstudie där flera olika metoder använts dels för materialinsamling, dels för att kunna jämföra resultatet av insamlat material med och mot varandra. Målet med tillvägagångssättet har varit att försöka återspegla en så korrekt bild av rådande förhållande i Kristianstad som möjligt.    Genomförd litteraturstudie av aktuell forskning och den nedskrivna praktiken hos  framgångsrika cykelstäder visar på en samstämmighet i  vilka kunskaper som finns i ämnet. Ingen vet exakt hur mycket olika strategier har påverkat en stads popularitet bland cyklister, men att kombinationen av ett antal åtgärder uppenbart gett resultat. Något som gör att kunniga kan resonera kring vad som troligen är betydelsefullt och vad som inte är det. För denna studie blir det dock en aning problematiskt när det inte finns några vetenskapligt belagda analysmetoder för en cykelnätsanalys, vilket gör att mer genomförande-inriktad litteratur gjort egna tolkningar där föreslagen analysmodell varierar. Hur Kristianstads cykelnät analyserats baseras därför på kunskap om vad man tror kan vara relevant att inkludera i analysen utifrån ett pendlarperspektiv. Ur analysen av de fysiska förutsättningarna för cykelpendlare hittades ett antal företeelser som kan anses bristfälligt: stadens glesa struktur, att trafiksäkerheten på ett antal platser är högst måttlig eller låg och att det på vissa sträckor kan vara svårt för pendlare att hålla en jämn och hög hastighet. I det avslutande kapitlet diskuteras cykelnätets potential för ökad cykelpendling  och där ett antal stråk föreslås särskilt anpassas för lite längre och snabbare cykelresor. Något som tillsammans med andra åtgärder, såsom att marknadsföra cykling eller göra det kostsammare att köra bil inne i staden, skulle kunna uppmuntra till ett ökat cykelpendlande i Kristianstad.

In this study, the homodimeric and heteromeric nature of ProP, an H+/solute symporter, from E. coli was analyzed. The measured initial rates of proline uptake via ProP-His6 and His6-ProP indicated that as the growth medium osmolality increased, the assay medium osmolality required for half maximal transport activity (Π½/RT) increased and the maximal uptake rate (Amax) decreased. The oligomeric state of ProP, as determined by Blue Native PAGE, showed that both monomeric and dimeric forms of the transporter were present in wild type and cardiolipin deficient bacteria expressing ProP, ProP-His6 or His6-ProP, after culturing in low or high growth medium osmolality. The BACTH System was used to confirm the homodimeric ProP-ProP interaction and to verify the heteromeric interaction between ProP and YdhP. Initial rates of proline uptake via ProP and Western blots indicated that replacement of the ydhP locus with a kanamycin cassette had no effect on ProP function or expression.

University, Diss., 2006--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

Regensburg, Univ., Diss., 2009.

University, Diss., 2004--Konstanz. / Erscheinungsjahr an der Haupttitelstelle: 2003.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Simona Dudičová Supervisor: Assoc. Prof. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: The effect of in vitro and ex vivo placental cells stimulation on expression of selected ABC and OATP transporters The placenta is an organ that plays a key role throughout pregnancy for proper fetal development. One of the important functions provided by the placenta is the transport of substances between the mother and the fetus. This transfer of substances enabled mainly by membrane transporters, which are located on the apical and basal membranes of the syncytiotrophoblast. During various physiological or pathological changes in the human body, their expression and amount can vary significantly. Inflammatory reactions that may occur during pregnancy are also related to these changes, and therefore we have addressed this issue and believed that this condition may alter the expression of placental transporters. The aim of this work was to investigate the changes in the expression of membrane transporters using placental cells on BeWo cell lines and placental villous explants that were stimulated by pro-inflammatory mediators. The change in the expression of individual ATP-binding cassettes,...

Avian coccidiosis is caused by the intestinal protozoa Eimeria. The parasite's site of infection in the intestine is site specific. Eimeria acervulina mainly affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain in Eimeria-challenged chickens. My previous studies showed that the growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. This can also lead to diminished intracellular pools of nutrients and inhibit pathogen replication. In this dissertation, further analysis of cellular events was performed. Expression of host defense peptides (HDPs), apoptosis and autophagy related genes were examined in Eimeria challenged broilers. The results showed that upon Eimeria infection, LEAP2 was consistently downregulated in the target tissues, while the avian beta-defensins (AvBDs) showed many variations in expression patterns. Downregulation of LEAP2 may be a mechanism for Eimeria to combat the host defense system, and to promote its survival inside the host cell. The in situ hybridization results showed that LEAP2 was expressed only along the villus in the small intestine and not in the crypt. This is the first time LEAP2 has been localized to epithelial cells of the chicken intestine. Eimeria infection can also induce an anti-apoptotic and anti-autophagy state in the host cells. This condition can be both favorable and unfavorable to parasite survival and replication inside the host cell. A comparison of gene expression between Ross and Eimeria resistant Fayoumi (line M5.1 and M15.2) chickens challenged with Eimeria maxima was conducted. The comparison among different lines of chickens showed differential gene expression patterns in lines with different resistance to Eimeria. The similar body weight reduction indicated that there may not be a significant Eimeria resistant line among the Ross, Fayoumi M5.1 and M15.2 birds. The interaction between Eimeria and the host cell is very complex. Studying the mechanisms behind the changes of gene expression during Eimeria infection may give rise to potential therapeutic targets of coccidiosis. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / The serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function. To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5- HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase (CAMKII). Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs.