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Diss. (sammanfattning) Uppsala : Universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Georgia State University, 2008. / Title from file title page. Dabney W. Dixon, committee chair; Kathryn B. Grant, Jerry Smith, committee members. Electronic text (171 p. ; ill. (some col.)) : digital, PDF file. Description based on contents viewed June 23, 2008. Includes bibliographical references.

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Heart disease is leading cause of death in the United States. High-density lipoprotein (HDL) levels are inversely correlated with the prevalence of coronary heart disease. The anti-atherogenic properties of HDL are associated with its role in the pathway of the reverse cholesterol transport, which removes cholesterol from peripheral cells for transport back to hepatocytes. The formation of HDL is facilitated by ATP cassette transporter ABCA1 and apolipoprotein A-I, the major protein of HDL particles. However, the underlying molecular mechanism behind the biogenesis of HDL is not well understood. To provide further understanding of this mechanism, we developed two ABCA1 expression systems in both Sf9 insect cells and FreestyleTM 293-F human cells for functional and structural studies. We designed all constructs of ABCA1 to contain a C-terminal rho1d4 tag that bound to an affinity matrix of rho1d4 antibodies for successful purification. To reconstitute ABCA1 in a detergent-free environment that models the native membrane, we developed three reconstitution systems for ABCA1: saposin A nanodiscs, peptidisc, and amphipol A8-35. Biochemical and structural studies were carried out to understand the mechanism behind ABCA1’s function. We demonstrated a potential direct interaction of ABCA1 and apolipoprotein A-I with a pull-down experiment. Two cryo-electron microscopy data collections were obtained of ABCA1 in a detergent environment in the presence of ATP with the goal of determining the structure of ABCA1’s active state. We produced a 12 Å reconstruction of ABCA1 from this first data collection. This low-resolution structure confirmed the general structure that currently exists for ABCA1. Processing the data helped us streamline and troubleshoot the electron microscopy workflow pipeline for future data collections. Unfortunately, the second data collection had astigmatism issues that prevented particle alignment during data analysis. However, these data collections provided considerable insight into the ideal sample freezing and grid preparation conditions that affect data collection and data processing. More transmembrane protein structures are being solved each year but there remain many obstacles and challenges in ABCA1 purification and grid preparation that affect the ability to perform functional studies and high-resolution structure determination. Our developmental work has helped move forward our biochemical understanding of ABCA1 to achieve these aims. The more that is learned about this important membrane protein the more likely it is that future consistent production of ABCA1 will be accomplished to answer the questions of how ABCA1 mediates the formation of HDL particles.

PepT1, PepT2 and PHT1 are all members of the proton-coupled oligopeptide transporter family, which are important in the transport of amino acids in peptide form. PepT1 acts as a low affinity/high capacity transporter and PepT2 as a high affinity/low capacity transporter for di- and tri-peptides. PHT1 transports di- and tri-peptides as well as histidine. The objective of this study was to profile PepT1, PepT2 and PHT1 mRNA expression in the proventriculus, duodenum, jejunum, ileum, ceca, large intestine, brain, heart, bursa of Fabricius, lung, kidney, and liver in layer chicks on embryonic days 18 and 20 and days 1, 3, 7, 10, and 14 post-hatch. Absolute quantification real-time PCR was used to measure gene expression. PepT1 expression was greatest in the duodenum, jejunum and ileum. Over time, PepT1 expression increased in the duodenum, jejunum, ileum and large intestine and decreased in the ceca. PepT2 expression was greatest in the brain, aiding in neuropeptide homeostasis, and the kidney, aiding in the reabsorption of substrates. Over time, PepT2 expression increased in the bursa of Fabricius and decreased in the proventriculus, duodenum, jejunum and liver. In the small intestine during embryogenesis, PepT2 may function to transport di- and tri-peptides prior to the induction of PepT1. PHT1 expression was expressed in all tissues analyzed. Over time, PHT1 expression increased in the jejunum, large intestine, brain and liver and decreased in the proventriculus. The uptake of peptides in the developing chick is regulated by peptide transporters that are expressed in a tissue- and development-specific manner. / Master of Science

Salmonella and Campylobacter are the leading causes of bacterial foodborne illness in the United States. Commonly found in the gastrointestinal tract of poultry, Salmonella and Campylobacter may show little to no signs of infection in birds. The objective of this dissertation was to evaluate the influence on mRNA abundance of nutrient transporters and host defense peptides (HDPs) during a Salmonella or a Campylobacter challenge in young commercial broilers. Comparisons were made between non-challenged and challenged (106, 107, or 108 colony forming units of Salmonella or Campylobacter) broilers on expression of nutrient transporters and host defense peptides in the duodenum, jejunum, ileum and cecum at various days after inoculation. During a Salmonella challenge, changes in mRNA abundance of nutrient transporters and avian beta-defensins (AvBD) vary by day, tissue and challenge dose. ZnT1 may play an important role during a Salmonella challenge as mRNA abundance of ZnT1 significantly increased (P<0.05) by day 7 in the 108 group compared to the control. Early changes in LEAP2 mRNA abundance were observed in the 106 group than the 107 and 108 groups. However, at a later time point post challenge, a lower abundance of almost all AvBD mRNA (P<0.05) was observed in the lower gastrointestinal tract especially in the 107 and 108 groups compared to the control group, indicating that the pathogen may be influencing intestinal expression of AvBD mRNA. In Campylobacter, analyses revealed that expression of zinc transporter 1 (ZnT1) increased (P<0.05) in the duodenum, ileum and ceca in the 106 group on day 7. An increase (P<0.05) in the expression of avian beta-defensins were observed on day 14 in the ileum and ceca in the 106 group compared to the control group. Pathogens like Salmonella and Campylobacter may have an influence on the mRNA abundance of nutrient transporters and HDPs. Manipulation of these genes may ensure the survivability of these pathogens. Through sequestration of nutrients, the pathogen would have the ability to colonize the host and replicate. However, it must evade the host immune system as well. The processing of infected poultry with these pathogens may lead to foodborne illness in humans. Further research is needed to investigate possible methods to counter the influence these pathogens have on host immunity genes. / Ph. D.

The nasal route has primarily been used to deliver drugs for the treatment of local diseases such as nasal infections, nasal congestion and allergies. The nasal route can also be used as a non-invasive alternative route to deliver drugs systemically when a rapid onset of action and/or avoidance of hepatic metabolism are desired. Moreover, there is a growing interest in the use of this route for direct transport of drugs from the nose to the brain. Most of the drugs that have been studied for nasal delivery are either small molecules which are lipophilic enough to passively diffuse through the nasal epithelia or macromolecules where bioavailabilities less than 1% are clinically effective and acceptable. This study focused on identifying carrier proteins or transporters in the nasal mucosa that could improve the absorption of specific drug substrates across the nasal respiratory and olfactory epithelia. The presence of drug transporters in the nasal mucosa of humans and commonly used animal models were investigated. DNA microarray results for nasal samples from humans and two commonly used models, mice and rats, were obtained from GenBank and were analyzed in collaboration with the University of Iowa Center for Bioinformatics and Computational Biology. While cow tissues are frequently used in in-vitro nasal permeability analyses, there is limited information available in GenBank for this species. Both DNA microarray analysis and RT-PCR were performed on bovine nasal explants to determine transporter expression. Good agreement between the microarray and RT-PCR results was observed. While human and three animal species commonly used as models in nasal drug delivery research (mouse, rat, and cow) show similar patterns of expression for several transporters, interspecies differences in the level of expression were observed. Therefore, the expression level of transporters remains a factor to consider when translating results obtained using animal models to humans. The nucleoside transporter family was selected for further evaluation of the potential to improve the nasal absorption of substrates. Nucleoside transporters are integral proteins responsible for mediating and facilitating the flux of nucleosides across cellular membranes; they are also known to be responsible for the uptake of nucleoside analog drugs such as anti-cancer and anti-viral agents. RT-PCR and Western blotting were used to verify the presence of two transporter subtypes, ENT1 and CNT3, in the bovine nasal respiratory and olfactory mucosa. The expression level of both transporters in the respiratory mucosa was comparable to that in the olfactory mucosa. Using immunohistochemistry, ENT1 and CNT3 were found to be localized primarily at the apical surface of the nasal epithelial cells. This indicates that the nasal epithelium likely absorbs exogenous nucleosides for intracellular uses such as nucleic acid synthesis and regulating other cellular activities. The contribution of the nucleoside transporters to the permeation of a nucleoside analogue drug, alovudine, across the nasal epithelia was also studied. The transport of alovudine showed a non-linear increase with increasing donor concentration over the range of 50 to 3000 µM which suggests that nucleoside transporters play a role in its uptake. Polarized transport was not observed suggesting that the facilitative nature of ENT1 plays a major role in alovudine transport. S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT1 inhibitor, incompletely decreased alovudine permeability across the nasal mucosa. This demonstrates that at least one transporter, ENT, plays a significant role in the uptake of this nucleoside drug across the nasal mucosa.

The emergence of multidrug resistance is a serious medical problem that has significantly affected the treatment of tumor cells and infectious diseases. This multidrug resistance phenotype is mediated by the action of a large family of membrane proteins that act as active transporters or energy driven efflux pumps in both of prokaryotic and eukaryotic cells. Most eukaryotic multidrug efflux pumps belong to the ATP binding cassette (ABC) family of transport proteins that include P-glycoprotein (P-gp1), Multidrug Resistance Associated Protein (MRP1), and Breast Cancer Resistance Protein (BCRP). In prokaryotic cells, Lactococcus lactis LmrA, a homolog of P-gp1, mediates drug resistance to antibiotics and cytotoxic drugs. The transport function of these proteins is facilitated by the hydrolysis of ATP. However, the mechanism by which these proteins bind to, and are able to transport structurally dissimilar drugs across the cell membrane remains poorly understood. In this thesis we have attempted to characterize the interactions of various ABC transporters (MRP1, BCRP, and LmrA) with structurally diverse drugs, using a well characterized photoreactive drug analogue of Rhodamine 123, [125I] iodoaryl azido-rhodamine 123 (IAARh123). In the case of MRP1 interaction with Rhodamine 123, it was of interest to determine the nature of MRP1 drug interactions. In that study, our results show that CHAPS (1-[(3-cholamidopropyl) dimethylamino]-1-propansulfate) and Brij35 inhibited the photolabeling of MRP1 with IAARh123, and this interaction occurred outside the lipid bilayer. These results were unexpected in light of previous results with another ABC transporter which also binds to Rhodamine 123. Consequently, we show that non-toxic concentrations of CHAPS and Brij35 potentiate the toxicity of two MRP1 substrates, vincristine and etoposide (VP16). In the second chapter, we have used IAARh123 to demonstrate for the first time that the BCRP mediates drug resi