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Female mosquitoes lay eggs after the consuming a blood meal. The iron storage protein ferritin could be involved with iron movement among body tissues in insects. Mosquito ferritin is present in hemolymph (blood) and the messages for the heavy and light chain subunits contain secretion signal sequences. These subunits may be targeted to the secretory pathway. We found that Aedes aegypti larval cells (CCL-125) exposed to iron as ferric ammonium citrate (FAC) increased ferritin secretion in a dose-dependent manner. In order to study the secretory pathway of ferritin, we attempted to disrupt the Golgi by treating CCL-125 cells with brefeldin A (BFA) and monensin. Unexpectedly, neither BFA nor monensin inhibits iron-induced ferritin secretion. These data suggest that either CCL-125 cells are highly resistant to these agents or ferritin is secreted independently of the classical ER-Golgi pathway.

In this thesis five iron phthalocyanines (FePc’s), four of which having different electron withdrawing or electron donating substituents, were evaluated as 0₂ reduction reaction (ORR) catalysts. The experimental approach simulated a PEM fuel cell environment using both ex-situ electrochemical techniques and in situ fuel cell testing. The kinetic ORR parameters for the FePc species each adsorbed on a pyrolytic graphite WE were evaluated at four temperatures (20, 40, 60, 80°C) in a novel half cell using cyclic voltammetry (CV) and rotating disk electrode (RDE) voltammetry. Kinetic ORR parameters included the overall ORR electron transfer number, reaction rate constants, cathodic Tafel slopes, electron transfer numbers in the rate determining step, and electron transfer co-efficients. An increase in temperature from 60°C to 80°C showed a decrease in the overall electron transfer number observed for all four substituted FePc species. A mechanism was also proposed based on the experimental results. The RDE results were confirmed using a rotating ring disk electrode (RRDE). From these RRDE results, the fraction of H₂0₂ produced (XH₂₀₂) at the disk during the ORR was calculated. These XH₂₀₂ values agreed with the overall ORR electron transfer numbers from the RDE results obtained under the same conditions. lron(Il) 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25 — hexadecachioro 29H,31H-phthalocyanine (FePcCI₁₆)was down selected for further investigation as the most stable and active substituted FePc species for the ORR. Both FePcCl₁₆ and the unsubstituted FePc, were supported on carbon and made into catalyst inks for carbon fibre paper (CFP) electrode testing, and then evaluated using CV in the N₂ purged, followed by the air saturated, electrolytes, respectively. Finally, MEA’s for fuel cell testing were made using FePc species catalyst ink cathodes, and commercial Pt/C anodes. The MEA’s were tested using custom designed and built fuel cell hardware. Open circuit voltages, polarization curves, and power curves were recorded. Initial results indicated the FePcCI₁₆ MEA’s showed superior stability, higher open circuit voltages, as well as better polarization and power curves when compared to the unsubstituted FePc species. It was found that FePc species with strongly electron withdrawing substituents, such as FePcCl₁₆ showed the highest stability and greatest ORR activity. FePc species, including pyrolized FeNx/C analogues, show much promise as alternatives to Pt in PEMFC’s, as well as dye sensitized solar cells (DSSC’s).

Bacterial infection is a leading cause of morbidity and mortality arising from platelet transfusions (1, 2). Storage of platelet products at room temperature (20 to 24ºC) provides ideal conditions for bacterial proliferation (1, 3-6). Furthermore, platelets are stored in plasma containing bioavailable iron that bacteria require to survive (7). Thus we hypothesize that the inclusion of iron chelators will bind and remove iron, thereby inhibiting bacterial growth in both culture medium and platelet concentrates. Additionally, we hypothesize that residual red blood cells (RBCs) in platelet units may contribute bioavailable iron that promotes bacterial growth. To test these hypotheses, we first assessed growth of Staphylococcus epidermidis in culture medium after treatment with the iron chelators deferoxamine (DFO) or phytic acid. DFO significantly inhibited bacterial growth in a dose dependent manner (p < 0.009). Conversely, phytate only inhibited bacterial growth at concentrations ≥ 100 mM (p < 0.001); at ≤ 5 mM, phytate supplied S. epidermidis with additional nutrients and significantly promoted growth (p < 0.001). Subsequently, we monitored the change in RBCs over time. Hemolysis, methemoglobin, and iron levels all significantly increased over the 7-day storage period (p < 0.001) releasing bioavailable iron. Indeed, we found that S. epidermidis growth in iron-poor medium drastically increased with the addition of RBCs, thus supporting our second hypothesis. Surprisingly, the inclusion of DFO in minimal medium did not demonstrate a bacteriostatic effect in the presence of RBCs. The inhibitory effect of DFO was likely overcome by iron released from the elevated methemoglobin levels arising from the direct interaction of DFO with hemoglobin. Previous studies demonstrate that methemoglobin releases iron more quickly than normal hemoglobin (8). Lastly, we evaluated the effect of DFO on microbial growth in platelet concentrates using the BacT/ALERT system. The presence of DFO significantly inhibited S. epidermidis growth in buffy coat platelets in a dose dependent manner (p < 0.001). With these findings, the inclusion of iron chelators is a promising approach to preventing transfusion-transmitted bacterial infection and providing patients with a safer platelet product.

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of harmful hydroxyl-free radicals. Ceruloplasmin is the major multicopper ferroxidase in blood; however, hephaestin, a membrane-bound ceruloplasmin homolog, has been implicated in the export of iron from duodenal enterocytes into blood. These ferroxidases supply transferrin, the iron-carrier protein in plasma, with Fe(III). Transferrin circulates through blood and delivers iron to cells via the transferrin receptor pathway. Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between the ferroxidase and transferrin. In Chapter 3, the putative interaction of transferrin with ceruloplasmin and a soluble form of recombinant hephaestin was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance, a stable interaction between the two proteins was not detected. The lack of interaction between hephaestin and transferrin prompted the investigation into the localization of hephaestin in the human small intestine. Hephaestin has been reported to have both intracellular and extracellular locations in murine tissue. In the Appendix, the location of hephaestin in human tissue was investigated using a novel polyclonal antibody. Hephaestin was localized to the basolateral membrane and an intracellular location of the enterocyte, as well as a novel location in the myenteric plexus of the duodenum. The delivery of iron to cells via the transferrin receptor pathway is well established; however, little is known about the interaction of transferrin with the transferrin receptor at the molecular level. In Chapters 4 and 5, surface plasmon resonance was employed to further characterize the binding event between transferrin and the transferrin receptor. It was found that mutations affecting iron release in transferrin did not impact receptor binding. However, when N-lobe residues predicted to form contacts with the transferrin receptor were targeted, significant changes in the transferrin receptor binding kinetics and affinity were observed.

Fresh and aged iron particle attachment onto model mineral surfaces was studied using atomic force microscopy based – force spectroscopy (AFM) in different water chemistries as well as in the presence of surface modifiers for the particles (carboxymethyl cellulose polymer). The forces of interactions measured with AFM were explained in terms of classical DLVO forces (electrostatic, Born and van der Waals forces) as well as hydration, steric and magnetic forces, which are not accounted for by classical models. Theoretical interpretation of the results was thus performed using extended models, in which such interactions were considered. In the absence of surface modifiers, iron particle attachment onto silicates and iron oxides appeared favorable in pure water and salt solutions at pH 4 and 5.5, but was hindered in water amended with humic acids. Attachment of bare CIP was also unfavourable at pH 8, except in the presence of CaCl2 salt at 100 mM concentrations. Attachment between iron particles and iron oxides was favorable even in the presence of polymeric coatings. Conversely, in all solutions such coatings contributed to electro-steric repulsion between particles and silicates, which dominated over attractive van der Waals forces, albeit to a lesser extent in the presence of salts. To best assess the role of polymeric coatings in particle attachment, polymer sorption onto the model mineral surfaces considered was investigated using a Quartz Crystal Microbalance with Dissipation Monitoring. The results obtained confirmed the AFM findings, and furnished insights regarding the role of salts on polymer sorption onto substrates. Finally, the effect of aging on attachment was probed both by AFM and by analyzing cementation phenomena of iron aggregates using scanning electron microscopy, as well as by probing changes in particle compositional properties with various spectroscopy tools. The major effect of aging was to promote the formation of large cemented aggregates, which did not appear prone to disruption or remobilization.