The obligate intracellular bacterium Chlamydia trachomatis requires iron in order to complete its developmental cycle. Addition of an iron-chelating drug, Desferal (deferoxamine mesylate), to infected cell culture causes Chlamydia to enter persistence. Here, we explore the ability of a stably-transfected cell line with inducible over-expression of the eukaryotic iron efflux protein ferroportin to starve C. trachomatis serovar E for iron. Ferroportin-induced iron removal is perhaps a more direct method of removing iron from the intracellular compartment versus exposure to an exogenous chemical chelator. Following induction, ferroportin-green fluorescent protein (Fpn-GFP) was detected in the plasma membrane, and cells expressing Fpn-GFP remained viable throughout the timescale required for Chlamydia to complete its developmental cycle. Following Fpn-GFP induction in infected cells, chlamydial infectivity remained unchanged, indicating chlamydiae were not in persistence. Ferritin levels indicate only a small decrease in cellular iron following Fpn-GFP expression relative to cultures exposed to Desferal. These data indicate that expression of Fpn-GFP in chlamydiae-infected cells is not capable of reducing iron below the threshold concentration needed to cause chlamydiae to enter persistence.

Release Date: October 2017 Anemia of chronic inflammation (ACI) is a condition that develops in a setting of chronic immune activation. ACI is characterized and triggered by inflammatory cytokines and the disruption of iron homeostasis. Hepcidin, a small peptide hormone, is the master regulator of iron homeostasis, and rapidly responds to iron supply and demand. Under conditions of chronic inflammation, hepcidin is elevated, and alters the way that iron is absorbed and disrupted throughout the body, resulting in disrupted iron homeostasis through inhibition of the iron exporter protein ferroportin. Anemia arises when insufficient erythropoietic activity combined with inadequate iron supply abrogates the healthy development of mature red blood cells (RBCs). The proprotein convertase (PC) known as furin is a serine protease capable of cleaving peptide precursors into their active state. Furin is critical for normal activation of proteins and enzymes but recently, furin has been implicated in critical roles within cancers, viral and pathogenic infections, and arthritis through activating precursors novel to the disease type. Furin has previously been identified as being the sole PC responsible for generating active hepcidin. Hepcidin is initially synthesized as a larger precursor protein, before undergoing furin cleavage. Furin is known to form mature, bioactive hepcidin, with the removal of this pro-region. Our discovery of a novel regulatory site on Furin has led to potent inhibition using small molecules. This inhibition is shown with the use of in vitro fluorogenic assays, in vivo cell tissue cultures, and within an animal model of ACI. Our results demonstrate that in using these small molecules we can decrease hepcidin levels even in the presence of potent inflammatory cytokines. The inhibition of hepcidin by these small molecules causes an increase in stably expressed ferroportin on cell surfaces and the restoration of the ability to mobilize iron from storage tissues and absorption from the diet. The ability to "fine-tune" inhibition of furin in targeting its allosteric site along with its catalytic domain designates these small-molecule inhibitors as promising therapeutics for treatment of diseases ranging from Alzheimer's and cancer to anthrax and Ebola fever.

Active learning strategies are important for facilitating deep learning that may be carried throughout life, but which is still finding its way into the college setting. Educators are not often trained in effective learning practices, which reduces the cognitive and proficiency gains of their students. By providing such guidance in the formative years of a teacher’s training, we hypothesize that the learning environment will be greatly enriched and enhanced. On the opposite end of the spectrum of life and cognition, the plague of dementia also warrants examination. Alzheimer’s disease (AD), an incurable neurodegenerative disorder progressing from the medial temporal lobe, is the most common form of dementia diagnosed in people over age 65, afflicting 30-40% of those 85 years and older. Despite its prevalence, effective treatments are limited because the principal causes and triggers of AD are not entirely understood. Growing evidence demonstrates that oxidative stress (OS) is an important factor contributing to the initiation and progression of AD. A key player contributing to this OS is iron, an essential trace mineral which is required for proper neuronal function, but which generates reactive oxygen species during redox transitions. Intracellular labile iron pool (LIP) levels are strictly regulated by proteins such as transferrin (import), ferroportin (export), and ferritin (storage). However, when these proteins become dysregulated, excess iron associates with other proteins such as amyloid beta (Aβ) and tau, aggregations of which are hallmarks of AD. In our hypothetical model, under extensive or prolonged OS, as occurs in AD, much larger Aβ plaques form because the stress does not abate. Hyperphosphorylated tau is the last resort to protect the cell against free iron, and aggregates when the LIP is elevated because neither iron storage in ferritin nor iron export through ferroportin can relieve the neurons of the free iron.

Iron is an essential nutrient for energy and DNA replication. Its homeostasis is commonly perturbed by chronic inflammatory mechanisms. Chronic inflammation upregulates a cytokine, hepcidin, that degrades the iron export protein ferroportin. Without a way to export iron into the bloodstream iron availability in blood becomes depleted. Iron depletion in the blood stream hinders erythropoiesis and is termed anemia. Herein I investigate and inhibit the mechanism of hepcidin activation. Inhibition of hepcidin activation has released iron from tissues and alleviated anemic conditions in a cancer model. I have laid the foundation to investigate this pathway in a 3D spheroid model. The results show that hepcidin-25 inhibition is a promising treatment for anemia of cancer. More work needs to be done to confirm efficacy in an in vivo model. In addition to anemia of cancer I have also worked with diabetic rats and investigated their anemic state using common anemia diagnostic methods. I found that in this high fat high sugar diet Wistar rat model anemia was not induced. In addition to my studies on anemia I have investigated the use of portable x-ray fluorescence (pXRF) as an accessible and affordable elemental analysis technique for lateral flow immunoassays and biological samples such as cell lysates and animal tissue. While pXRF shows promising results more work needs to be done to increase its sensitivity and pixel size.

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[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. In mammals since no controlled means of eliminating unwanted iron has evolved, body iron balance is maintained by alterations in dietary iron intake. This occurs in the duodenum where most dietary iron is absorbed. Absorption involves at least two steps, uptake of iron from the intestinal lumen and then its transport into the body, processes that occur at the apical and basal membranes of enterocytes, respectively. In chapter one of this thesis the background information relevant to iron absorption is described. Despite numerous studies, the role of these proteins in iron absorption remains unclear, partly because many studies have reported them in non-enterocyte cell lines where the expression of the proteins involved in iron absorption is unlikely and therefore the physiological significance of the findings uncertain. Therefore, the study of iron absorption would value from additional cell lines of intestinal origin being used, preferably derived from a species used to comprehensively study this process in vivo, namely the rat. Validation of such a model would enable comparisons to be made from a molecular level to its relevance in the whole organism. In chapter 3 of this thesis, the rat intestinal cell line 6 (IEC-6) was examined as a model of intestinal iron transport. IEC-6 cells expressed many of the proteins involved in iron absorption, but not the ferrireductase Dcytb, sucrase or αvβ3 integrin. In addition, in IEC-6 cells the expression of the apical transporter divalent metal transporter 1 (DMT1), the iron storage protein ferritin, the uptake of Fe(II) and Fe(III) were regulated by cellular iron stores as is seen in vivo. This suggests that IEC-6 cells are of a lower villus enterocyte phenotype. Presented in chapter 4 is the study of the uptake of iron from Fe(II):ascorbate and Fe(III):citrate by IEC-6 cells in the presence of a blocking antibody to the putative basolateral transporter ferroportin1 and of colchicine and vinblastine, different pHs, and over-expression of DMT1. It was shown that optimal Fe(II) uptake required a low extracellular pH and was dependent on DMT1. Uptake of Fe(III) functioned optimally at a neutral pH, did not require surface ferrireduction, and was increased during over-expression of DMT1. These observations suggest that intravesicular ferrireduction takes place before transport of Fe(II) to the cytoplasm by DMT1. This pathway was not blocked by a functional antibody against αvβ3 integrin but was inhibited by competition with unlabeled iron citrate or citrate alone. Surprisingly, a functional antibody against ferroportin1 had no effect on efflux but significantly reduced (p<0.05) uptake of Fe(II) by 40-50% and Fe(III) by 90%, indicating two separate pathways for the uptake of iron from Fe(II)-ascorbate and from Fe(III)-citrate in IEC-6 cells. Presented in chapter 5 is the development and validation of a technique for the removal of freshly isolated enterocytes from the rat duodenum and their use to study iron transport processes that enabled comparisons to be made between these cells, IEC-6 cells and the human enterocyte cell line Caco-2 cells. In chapter 6 a blocking antibody to ferroportin1 was shown to inhibit uptake of Fe(II) but not release of iron in freshly isolated duodenal enterocytes from rats and Caco-2 cells supporting the findings obtained with IEC-6 cells described in chapter 4. Fe(II) uptake was reduced only when the antibody was in contact with the apical membrane indicating its expression at the microvillus membrane. Confirming this, ferroportin1 was shown along the microvillus membrane of Caco-2 cells, in enriched microvillus membrane preparations and in enterocytes of duodenum tissue of rats where it co-localised with lactase. The significant findings to emerge from this thesis are that the IEC-6 cell is a valid model to study iron absorption producing results consistent with those found in freshly isolated enterocytes and in human enterocyte-like cells. In particular, ferroportin1 functions in the uptake of iron at the apical membrane possibly by modulating surface binding of Fe(II) to DMT1 or the activity of DMT1. In addition to this in Fe(II) uptake from Fe(III) ferroportin1 may also affect the number of Fe(III): citrate binding sites. Preliminary studies further characterizing the function of ferroportin1 at the apical membrane and at intracellular sites of IEC-6 cells along with integration of these data are discussed in chapter 7.

Le fer est un oligoélément indispensable pour tout organisme vivant. Le taux de fer systémique est régulé par la fixation de l’hepcidine, hormone synthétisée majoritairement par le foie mais également par les macrophages, à la ferroportine seul exporteur du fer. L’expression de ces deux protéines est régulée par le taux de fer et les processus inflammatoires. Des mécanismes d’acquisition et de séquestration du fer sont mis en place respectivement par le pathogène et l’hôte durant l’infection et régulent en parallèle l’expression de l’hepcidine et la ferroportine. Les travaux de recherche effectués dans le cadre de ma thèse ont porté d’une part sur un aspect fondamental à améliorer nos connaissances du mécanisme de régulation de l’axe hepcidine - ferroportine en condition inflammatoire et analyser l’influence du fer sur la réponse immune au niveau des macrophages; d’autre part une deuxième partie de mes recherches s’est orientée vers une étude plus appliquée du rôle du fer dans la réponse immune induite par une infection mycobactérienne. Nous montrons que l’expression de l’hepcidine et de la ferroportine est différentiellement régulée en corrélation avec la polarisation des macrophages via les voies de signalisation intracellulaires PI3K et autres kinases. Le fer influence la polarisation des macrophages et module ainsi la réponse inflammatoire, et représente aussi un signal de danger capable de stimuler une voie MyD88-dépendante. Enfin, la réponse à l’infection Mycobacterium. bovis BCG est modulée par un régime modérément enrichi en fer, réduisant la charge bactérienne et l’inflammation. / Iron is an essential trace element for all organisms. In mammals, systemic iron homeostasis relies on hepcidin, a peptide hormone synthesized by liver but also macrophages with defensing properties, and its target, the cell iron exporter ferroportin. Iron content and inflammation regulate hepcidin and ferroportin expression in mammals. During infection, pathogens develop sophisticated mechanisms for iron acquisition and sequestration. In response, host regulates the bioavailability of iron through hepcidin and ferroportin expression. First, this work contributes to improve our fundamental knowledge on hepcidin and ferroportin regulation during inflammation and analyzes the influence of iron in macrophages immune response. Second, the role of iron in response to mycobacterial infection was investigated. We show that hepcidin and ferroportin expression was regulated differentially in correlation with macrophages polarization through intracellular signaling pathways involving PI3K and others kinases. In addition, iron influenced macrophages polarization leading to a decrease of inflammatory response with a potent effect on MyD88 pathway stimulation. Finally, we showed that moderate iron-rich diet modulated Mycobacterium bovis BCG response reducing the bacterial burden and inflammation.

Iron is an essential element for human organism, because it cooperates as a cofactor of enzymes in many metabolic pathways. Iron is a component of hemoglobin, and thus it is indispensable for the oxygen transport to tissues. It can exist as a ferrous or ferric form. However, ferrous iron paticipates in reactions in which highly reactive hydroxyl group can be formed. This product is harmful for the organism. Non-heme iron is taken up to the circulation through duodenal enterocyte. Iron excretion is carried out only by desquamation of the enterocytes or by bleeding. Therefore, iron intake must be strictly regulated. Iron overloading is observed in some chronic diseases (hereditary hemochromatosis, alcohol liver disease). In contrary, iron depletion can be a case of iron deficiency anemia. The aim of this master thesis is to determine the expression of iron transport molecules in duodenum in chronic diseases which originate due to disturbances of iron intake regulation. We determine the expression of molecules of iron transport (DMT1, Dcytb, ferroportin, hephaestin) on mRNA level by qPCR and on protein level by western blot. The level of serum hepcidin was determined by ELISA. Our results show an increased expression of mRNA of transporters DMT1 and ferroportin as well as ferrireductase Dcytb and ferroxidase...

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