<p>Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine <i>in vivo</i>. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human <i>jejunum</i> for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2. </p><p>The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.</p>

Cytochrome P450 enzymes (CYPs), found in almost all organisms, are involved in endobiotic metabolism and detoxification of xenobiotic compounds, such as drugs, pollutants, and insecticides. In insects, CYPs play a major role in conferring resistance to various insecticides including DDT. In Drosophila and other insects, DDT-resistant strains exhibit increased expression of multiple P450 genes; however, the mechanism of overexpression is unknown. Since many CYP genes including Cyp6a8 of Drosophila are induced by caffeine and other xenobiotics, these chemicals are used as tools to understand the regulation of these genes. Previously it was shown that the 0.8-kb (-1/-732) and 0.2-kb (-1/-170) upstream DNA of Cyp6a8 of the DDT-resistant 91-R strain support caffeine, DDT, and Phenobarbital induction in adult flies and S2 cells, the 0.2-kb DNA has many transcriptionally important sequence motifs. In the present investigation, site-directed mutagenesis was performed on the putative TATA box and CREB/AP-1 motifs located at the -97/-101, -57/-61, -43/-47, and -6/-10 regions of the 0.2- and 0.8 DNAs to determine their cis-regulatory role in caffeine and PB induction in S2 cells using luciferase reporter system. Results showed that all four deletions in 0.2- and 0.8-kb DNA decreased both basal and caffeine-induced activities, but maximum effect was seen with the -57/-61 deletion. Second, the TATA mutations greatly decreased basal activity, but they did not decrease caffeine-inducibility as much as the -57/-61 mutations. Third, the effects of other three deletions on basal activities were not as pronounced in the 0.8-kb environment as were seen in the 0.2-kb environment. Taken together these results suggest that of all four putative CREB/AP1 sites the one located at -57/-61 region is most important for both basal and caffeine-induced activities. The results also suggest that the additional 600 bases upstream of -1/-170 have distal elements that interact with the proximal promoter in the 0.2-kb DNA and boost basal transcription. A model suggesting interactions of all cis elements with the basal promoter for basal and induced transcription has been proposed.

Colon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously.

Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine in vivo. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human jejunum for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2. The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.

Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

<p>Excess cardiac myofibroblasts in fibrotic heart diseases as well as cell-based therapies involving implantation of stem cells or genetically engineered somatic cells in the heart may all lead to a situation where a cardiomyocyte becomes electrically coupled to an unexcitable cell. In these settings, electrotonic loading of cardiomyocytes by unexcitable cells can affect cardiac action potential generation, propagation, and repolarization depending on the properties of both cardiomyocytes and unexcitable cells. The objective of this dissertation was to advance our understanding of the electrical interactions between cardiomyocytes and unexcitable cells using a variety of electrophysiological, molecular, and cell culture techniques.</p><p>First, we utilized aligned cardiomyocyte monolayers covered with unexcitable cardiac fibroblasts or human embryonic kidney-293 (HEK) cells that expressed similar levels of the gap junction protein connexin-45. These cells weakly coupled to cardiomyocytes and marginally slowed cardiac conduction only at high coverage density, while producing no other measurable electrophysiological changes in cardiomyocytes. In contrast, unexcitable HEK cells genetically engineered to stably express the more conductive connexin-43 channels (Cx43 HEK) strongly coupled to cardiomyocytes, depolarized cardiac resting membrane potential, significantly slowed impulse propagation, decreased maximum capture rate, and increased action potential duration (APD) at high coverage density. None of the studied unexcitable cells significantly altered conduction velocity anisotropy ratio or the relatively low incidence of pacemaking activity of cardiac monolayers at any coverage density.</p><p>Next, we utilized individual micropatterned cell pairs consisting of a cardiomyocyte and an unexcitable Cx43 HEK cell with or without stably overexpressed inward rectifier potassium channels (Kir2.1+Cx43 HEK). By systematically varying the relative sizes of micropatterned cells, we showed that Cx43 HEK cells significantly depolarized cardiomyocytes, reduced maximum upstroke velocity and action potential amplitude, prolonged APD, and modulated beating rate as a function of HEK:CM area ratio. In contrast, in cell pairs formed between cardiomyocytes and Kir2.1+Cx43 HEK cells we observed significant reduction in cardiomyocyte action potential amplitude, duration, and maximum upstroke velocity, but no change in other measured parameters.</p><p>Finally, we utilized a hybrid dynamic clamp setting consisting of a live micropatterned cardiomyocyte coupled in real time to a virtual model of capacitive and/or ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells. We found that coupling of cardiomyocytes to the ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells was sufficient to reproduce the dependence of cardiomyocyte maximal diastolic potential and pacemaking behavior on HEK:CM area ratio observed in micropatterned cell pairs, but did not replicate the observed changes in action potential upstroke or duration. The pure capacitance model with no ionic current, on the other hand, significantly decreased cardiomyocyte maximum upstroke velocity and prolonged cardiomyocyte APD as function of HEK:CM area ratio without affecting maximal diastolic potential or pacemaking behavior. When the unexcitable cell model containing both capacitive and ionic currents was connected to cardiomyocytes, all changes in action potential shape observed in micropatterned cell pairs were accurately reproduced. </p><p>These studies describe how coupling of unexcitable cells to cardiomyocytes can alter cardiomyocyte electrophysiological properties dependent on the unexcitable cell connexin isoform expression, ion channel expression, and cell size. This knowledge is expected to aid in the design of safe and efficient cell and gene therapies for myocardial infarction, fibrotic heart disease, and cardiac arrhythmias.</p> / Dissertation

Colon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously.

Magnetic fluid hyperthermia is a potential therapy for achieving interstitial hyperthermia and is currently under clinical trials. This approach is based on the instillation of magnetic nanoparticles at the tumour site, which dissipate heat when exposed to an alternating magnetic field. This procedure leads to a local increase of temperature and induction of tumour death or regression. Nanoparticles of metallic iron are potential heating agents for this therapy, but rely on the presence of a protecting coat that avoids reactions with their environment. In this work, iron nanospheres and iron nanowires with a graphite coat are explored for this purpose. From these two nanostructures, the nanospheres are shown to have a greater potential in terms of heat dissipation. The graphite shell is further investigated as an interface for conjugation with other molecules of relevance such as drugs and fluorescent probes. The effect of acidic treatments on the magnetic and surface properties of the nanospheres is systematically studied and a suitable method to generate carboxylic functionalities on the nanoparticle surface alongside with a good preservation of the magnetic properties is developed. These carboxylic groups are shown to work as a bridge for conjugation with a model molecule, methylamine, as well as with a fluorescent dye, allowing the detection of the nanoparticles in cells by means of optical methods. The carboxylic functionalities are further explored for the conjugation with the anti-cancer drug cisplatin, where the amount of drug loaded per particle is found to be dependent on the density of free carboxylic groups. The release of the drug in physiological salt solutions is time and temperature dependent, making them particularly interesting for multi-modal anti-cancer therapies, where concomitant hyperthermia and chemotherapy could be achieved. Their potential for such therapies is shown in vitro by inducing hyperthermia in cell suspensions containing these nanoparticles. These results are finally translated to a three dimensional cell culture model where the in vitro growth of tumour spheroids is inhibited. The developed nanostructures have a great potential for therapeutic approaches based on the synergistic effects of hyperthermia and chemotherapy.

Ph.D.