Aspergillus fumigatus is a ubiquitous fungus associated with several airway complications and diseases including asthma, allergies, cystic fibrosis, and most commonly invasive aspergillosis. The airway epithelium, a protective barrier, is the first anatomical site to interact with A. fumigatus. Although this host-fungal interaction is often asymptomatic for immunocompetent individuals, for immunocompromised persons, due to a weakened competence of the immune system, they have an increased likelihood of fungal infection. This dissertation aims to investigate the effect of A. fumigatus on the transcriptional response of human airway epithelial cells, focusing on the relationship between innate immunity and iron regulation from the host perspective. The trace element iron is needed by both the fungus and the host for cellular maintenance and survival, but tightly controlled iron regulation in the host is required to prevent oxidative stress and cell death. The research methods in this dissertation employ a systems biology approach, by incorporating mathematical modeling, RNA-seq analysis, and experimental biology techniques to assess the role of airway epithelial cells in the host-fungal interaction. Both the quantitative and qualitative research design allows for characterization of airway epithelial cells and the downstream changes in iron importer genes. This study addresses literature gaps through analysis of the host transcriptome using multiple time points, by performing an extensive evaluation of the effect of cytokines on iron importer genes, and conceptualization of a comprehensive mathematical model of the airway epithelial cell. The major findings suggest the following: 1) airway epithelial cells avidly respond to A. fumigatus through modification of the expression of immune response related genes at different infection stages, 2) during A. fumigatus co-incubation with airway epithelial cells, the iron importers genes respond in strikingly different ways, and 3) cytokines have a significant effect on the increase in expression of an iron importer gene. We illuminated the role of airway epithelial cells in fungal recognition and activation of the immune response in signaling cascades that consequently modify iron importer genes and hope to use this information as a platform to discover potential therapeutic targets. / Ph. D.

Methods were developed to isolate and culture the predominant cell types from porcine liver to investigate hepatic accumulation, distribution and intracellular metabolism of supplemental iron. Hepatocytes were prepared from collagenase perfused livers by differential centrifugation, while Kupffer cells and endothelial cells were isolated by centrifugal elutriation. One day old piglets were injected with iron-dextran (Fe-dextran) and the concentration of accumulated iron was determined in all three cell types 1, 5, and 10 days later. The concentration of iron increased markedly in all three cell types when compared to cells isolated from untreated piglets (Kupffer cells > endothelial cells >> hepatocytes). Accumulated iron was subsequently mobilized from all three cell types. The role of ferritin in metabolism and storage of accumulated iron was investigated. An antiserum was prepared against porcine liver ferritin and the quantity of cellular ferritin was measured by immunoelectrophoresis. The amount of cellular iron associated with ferritin was assessed by ion exchange chromatography. All three types of liver cells accumulated ferritin in response to Fe-dextran treatment. Higher concentrations of ferritin-iron and ferritin-protein were present in Kupffer and endothelial cells than in hepatocytes at all times after iron treatment. However, at 1 day after treatment 48% of the total iron within hepatocytes was associated with ferritin; ferritin-iron accounted for only 10% of total cell iron by day 10. In contrast, ferritin-iron represented only approximately 9% of the total iron in sinusoidal cells throughout the study period. The possibility that accumulation of Fe-dextran enhanced peroxidation of membrane lipids was evaluated. Lipids extracted from heart and liver of iron-treated piglets contained increased levels of conjugated dienes. High levels of conjugated dienes were present in endothelial cells and hepatocytes 1 day after treatment and only in endothelial cells by day 5. Although Kupffer cells accumulated substantial quantities of Fe-dextran, conjugated dienes were not detectable. These studies indicate that treatment of piglets with Fe-dextran may selectively impair function of hepatic endothelial cells and perhaps hepatocytes, and define new criteria for evaluating compounds that are used for iron supplementation. / Ph. D. / incomplete_metadata

The effects of exercise training and iron intake on iron metabolism in adult female rats were investigated. Adult female Sprague-Dawley rats were assigned to either an exercise (E) or sedentary (S) group and fed either a diet containing 9 ppm (9) of dietary iron (low iron level), or 40 ppm (40) of iron (a level slightly above the National Research Council recommendations). The exercise animals were subjected to a program of swimming, 5 days/week, over a 6- week period. Total food intake and final body weight were similar between the E and S groups. In both 40-E and 9-E animals, concentration of serum iron was significantly (P<0.05) lower while total iron binding capacity was significantly elevated, when compared to sedentary counterparts. Saturation of transferrin was significantly reduced in the 9-E group. Liver and spleen weights did not differ but significant increases in cardiac weights were noted in both E groups. Gastrocnemius muscle weights were similar in both E groups and 9-S, but significantly lower in the 40-S group. In organ tissues, liver iron concentration was significantly reduced in the 9-E animals, while spleen iron level was highest in the 40-E group. Cardiac iron concentration was significantly reduced in both E and low iron diet groups while levels of iron in gastrocnemius muscle did not differ among experimental groups. In both groups of exercised rats, bone marrow iron was significantly lower when compared to sedentary animals. In response to exercise training, an increase in skeletal muscle citrate synthase activity was observed in both E groups. This study suggests that exercise affects various parameters of iron metabolism. Regardless of iron intake, physical training appeared to alter distribution of iron stores, that may be associated with alterations of hematological iron transport and iron-containing proteins. The Combination of a low iron intake and intense exercise training appeared to enhance early characteristics of a latent iron deficiency. / Ph. D.

A presente pesquisa teve a finalidade de estudar a hematologia de bezerros clonados da raça Nelore, originados por meio da técnica de transferência nuclear de células somáticas (TNCS) por meio da avaliação do quadro eritrocitário, da dinâmica dos tipos de hemoglobina e do metabolismo do ferro destes animais durante o primeiro mês de vida. O delineamento experimental envolveu a colheita de 260 amostras de sangue e soro sanguíneo de 20 bezerros distribuídas nos seguintes momentos: imediatamente após o nascimento, 12 horas após o nascimento, 1 dia de vida, 2 , 3 , 4, 5, 7, 10, 15, 20 e 30 dias de vida. Os animais foram divididos em quatro grupos experimentais: 12 bezerros obtidos por meio da técnica de TNCS pelos laboratórios A e 8, 4 por meio de fertilização in vitro (FIV) e 4 por monta natural (MN). A ocorrência de anemia de grau moderado a grave, do tipo normocítico e normocrômico, foi observada em 100 % (5/5) dos 5 bezerros clonados pelo Laboratório A, enquanto a incidência nos bezerros clonados pelo Laboratório B foi igual a 14,2 % (1/7), nos bezerros obtidos por fertilização in vitro foi igual 50,0 % (2/4) e em bezerros obtidos por monta natural foi igual a 50,0 % (2/4). A avaliação do eritrograma dos bezerros cio nados pelo Laboratório A evidenciou que a anemia instalou-se gradualmente a partir das 12 horas de vida atingindo ao final da primeira semana, a, sua intensidade máxima, sendo observada a recuperação gradativa dos valores eritrograma a partir do 15°dia de vida. Os valores médios obtidos para o eritrograma dos bezerros clonados do Laboratório A no 7º dia de vida foram os seguintes: Hemácias - 4,33X106/mm3-; Volume Globular - 23 %, Taxa de Hemoglobina - 7,25 g/dL; VCM - 52,89 µ3-; HCM - 16,65 pg; CHCM - 31,47%. A anemia observada nos bezerros clonados pelo Laboratório A era de origem ferropriva, pois se evidenciou nesses animais uma significativa diminuição dos teores séricos de ferro associada à diminuição do índice de saturação da transferrina (1ST), enquanto os valores da capacidade total de ligação do ferro (CTLF) não sofreram influência durante o período. Os valores médios obtidos para o metabolismo de ferro dos bezerros clonados pelo Laboratório A no 7º dia de vida foram os seguintes: teores séricos de ferro - 47,35 mg/dL; capacidade total de ligação de ferro - 455,90 mg/dL, índice de saturação da transferrina - 9,64%. Durante o estudo dos tipos de hemoglobinas, utilizando-se técnica de eletroforese foram identificados três fenótipos de hemoblogina adulta (Hb-A; Hb-B e Hb-AB) e a presença de hemoglobina fetal (Hb-F), não sendo observadas anomalias que pudessem sugerir a ocorrência de hemoglobinopatias hereditárias e/ ou congênitas. Verificou-se que as taxas de Hb-A, nos clones com fenótipo Hb-AB e Hb-A, permaneceram estáveis durante todo o período experimental, enquanto nos bezerros obtidos por fertilização in vitro ou monta natural com os mesmos fenótipos (Hb-A e Hb-AB) observou-se a partir de 120 horas de vida um gradativo aumento das taxas de Hb-A. Durante a avaliação da dinâmica da hemoglobina do tipo fetal (Hb-F) no primeiro mês de vida observou-se, que todos os grupos animais apresentaram comportamento similar, caracterizado por sua diminuição com o desenvolvimento etário. / The present work aimed to study the hematology of cloned Nelore calves produced using the technique of somatic cell nuclear transfer (SCNT), by evaluating erythrocyte parameters, hemoglobin dynamics, and iron metabolism in the animals during the first month after birth. The experimental design included the collection of 260 blood and blood serum samples from 20 calves in the following times: immediately after birth, 12 hours after birth, 1st of life, 2nd, 3rd, 4th, 5th, 7th, 20th and 30th day of life. The animals were classified in four experimental groups: 12 calves produced, using SCNT for two commercial laboratories (laboratory A=5 calves and laboratory B= 7 calves), four calves produced by in vitro fertilization (IVF) and four calves produced by natural mating (NM). Mild to severe normocytic and normochromic anemia was observed in 100% (5/5) cloned calves from laboratory A, and 14.2% (117) cloned calves from laboratory B. In both IVF and NM calves, anemia was observed in 50% (214) of the calves. Erytrogram evaluation of cloned calves from laboratory A showed that anemia developed gradually from 12 hours after birth, was most intense at the end of the first week, and then erytrogram normal values were recovered after the 15th day of life. Mean values for the laboratory A cloned calves erytrogram in the 7th day of life were the following: Red cells 4033X106/mm3, hematocrit 23%, hemoglobin 7.25g/dL, MCV 52.89µ3-; MCH 16.65, MCHC 31.47%. Anemia observed in cloned calves from laboratory A was caused by iron deficiency, since a significant decrease in iron se rum levels together with a decrease in transferrin saturation index (TSI) was confirmed. At the same time, Total iron-binding capacity (TIBC) was not changed in this period of time. Mean iron metabolism values for cloned calves from laboratory A were the following: serum iron amount: 47,35mg/dL, TIBC 455,90 mg/dL and TSI 9,64%. Hemoglobin identification by eletrophoresis identified three adult hemolglobin phenotypes (A-Hb; B-Hb e AB-Hb) and the fetal hemoglobin (F-HB), and there was no sign of hereditary hemoglobin disorders were observed. The rate of A-Hb in cloned calves with A-Hb and AB-Hb phenotypes was maintained during the experimental period. Nonetheless, for the IVF calves with the same phenotypes (A-Hb and AB-Hb) a constant increase in the A-Hb was noticed. For the evaluation of F-Hb dynamics in the first month of life, results showed similar pattern, characterized by its decrease with age.

Les Iron Regulatory Proteins 1 (IRP1/2) sont des protéines cytosoliques qui contrôlent l’homéostasie du fer chez les mammifères. Elles régulent la concentration de fer intracellulaire au niveau post-transcriptionnel, en interagissant spécifiquement avec des motifs appelés iron responsive élément (IREs). Ces motifs sont localisés dans les régions non traduites des ARNm codant notamment pour la ferritine (Ft), la ferroportine (Fpn) et le récepteur de la transferrine (TfR1). L’IRP1 est une protéine bifonctionnelle, majoritairement exprimée sous une forme contenant un centre [4Fe-4S] qui présente une activité aconitase. Les deux activités de l’IRP1 (aconitase/trans-régulateur) s’excluent mutuellement par la présence ou non du centre Fe-S. L’IRP2 est exprimée constitutivement sous une forme liant les IREs. Le monoxyde d’azote (NO), une importante molécule de signalisation impliquée dans les défenses immunitaires, cible le centre Fe-S de l’IRP1 et permet la conversion de l’IRP1 de sa forme aconitase vers sa forme liant les séquences IREs. Il a également été rapporté que l’IRP2 détecterait NO, cependant la fonction intrinsèque de l’IRP1 et de l’IRP2 dans le contrôle du métabolisme du fer intracellulaire en réponse à NO reste à ce jour non élucidée. Dans cette étude, nous avons identifié le régulateur principal du métabolisme du fer intracellulaire en réponse à NO, en utilisant des modèles de souris déficients pour les gènes IRP1 et/ou IRP2 et testé la contribution de la tension en oxygène dans cette régulation. Ainsi, nous avons exposé des macrophages primaires issus de la moelle osseuse de souris Irp1-/-, Irp2-/- et de souris Irp1-/- Irp2-/- de la lignée macrophagique à une source de NO, sous différentes tensions en oxygène. Les activités IRPs, l’expression des gènes Ft, Fpn et TfR1 ainsi que l’activité d’une protéine à centre Fe-S (l’aconitase mitochondriale) ont été mesurées après fractionnement cellulaire. Nous avons montré qu’en normoxie, la conversion de l’aconitase cytosolique en apo-IRP1 par NO est entièrement responsable de la régulation post-transcriptionnelle des ferritines (L-Ft et H-Ft), de la Fpn et du TfR1. En augmentant le transport du fer intracellulaire et en diminuant le stockage et l’export, l’activation de l’IRP1 par NO servirait à maintenir des taux de fer intracellulaire suffisants pour alimenter la biogenèse des centres Fe-S après l’arrêt des flux de NO. En effet, nous observons une restauration efficace de l’activité de l’aconitase mitochondriale dans les macrophages de souris sauvage alors qu’elle est bloquée dans les macrophages de souris Irp1-/-. De plus, l’IRP1 activée par NO, permet également de diminuer les taux de L- et H-Ft, anormalement élevée dans les macrophages de souris Irp2-/-. Nous montrons que le NO endogène active l’IRP1 sous sa forme trans-régulatrice alors qu’il tend à diminuer l’activité de l’IRP2. Néanmoins, l’IRP1 reste le régulateur principal des ferritines en conditions de normoxie. En condition hypoxique, les deux IRPs semble coopérer pour inhiber la traduction des ferritines car dans les macrophages Irp1-/-exposés à NO, l’IRP2 stabilisée est suffisante pour inhiber la traduction de la L- et H-Ft et ceci malgré l’activation transcriptionnelle des gènes de la L- et H-Ft. Concernant la régulation du TfR1 par NO et en hypoxie, TfR1 est principalement régulé par une voie transcriptionnelle dominant largement la voie post-transcriptionnelle impliquant l’IRP1. Le facteur de transcription HIF-1 alpha pourrait être le régulateur critique dans cette régulation. En conclusion, nous montrons dans cette étude, comment le regulon IRP participe à la régulation du métabolisme du fer intracellulaire en réponse à NO et son étroite connexion avec la concentration en oxygène. Nos résultats soulignent l’importance d’explorer davantage le rôle de l’IRP1 dans des situations inflammatoires in vivo, où les tissus peuvent être exposé à un microenvironnement non hypoxique. / Iron Regulatory Protein 1 (IRP1) and 2 (IRP2) are two cytosolic regulators of mammalian cellular iron homeostasis. IRPs post-transcriptionally modulate expression of iron-related genes by binding to specific sequences, called Iron Regulatory Elements (IREs), located in the untranslated regions (UTR) of mRNAs. Either of the two IRPs inhibits translation when bound to the single 5’UTR IRE in the mRNA encoding proteins of iron export (ferroportin - Fpn) and storage (ferritin - Ft) or prevents mRNA degradationwhen bound to the multiple IREs within the 3’UTR of the mRNA encoding the transferrinreceptor 1 (TfR1) - iron uptake molecule. The IRE-binding activity of both IRPs respondsto cellular iron levels, albeit via distinct mechanisms. IRP1 is a bifunctional protein, whichmostly exists in its non IRE-binding, [4Fe-4S] aconitase form and can be regulated by apost-translational incorporation or removal of the Fe-S cluster. In contrast to IRP1, IRP2 isnot able to ligate an Fe-S cluster, and its IRE-binding activity is determined by the rate ofits proteasomal degradation. Although both IRP1 and IRP2 can regulate cellular ironhomeostasis, only mice lacking IRP2 were shown to display iron mismanagement in mosttissues. This could be explained by the fact that IRP1 exists mostly in its non IRE−binding,aconitase form under physiological oxygen conditions (3-6%). Interestingly, nitric oxide(NO), an important signalling molecule involved in immune defence, targets the Fe-Scluster of IRP1 in both normoxia and hypoxia, and converts IRP1 from aconitase to anIRE-binding form. It has also been reported that IRP2 could sense NO, but the intrinsicfunction of IRP1 and IRP2 in NO−mediated regulation of cellular iron metabolism hasremained a matter of controversy. In this study, we took advantage of mouse models ofIRP deficiency to define the respective role of IRP1 and IRP2 in the regulation of cellulariron metabolism by NO and assess the contribution of oxygen tension on the regulation.Therefore, we exposed bone marrow-derived macrophages (BMMs) from Irp1-/-, Irp2-/- andmacrophage specific double knockout mosaic mice (Irp1/2-/-) to exogenous andendogenous NO under different oxygen conditions (21% O2 for normoxia and 3-5% forhypoxia experiments) and measured IRPs activities, iron-related genes expression andactivity of Fe-S cluster protein – mitochondrial aconitase. We showed that in normoxia, thegenerated apo-form of IRP1 by NO was entirely responsible for the post-transcriptionalregulation of TfR1, H-Ft, L-Ft and Fpn. Moreover, by increasing iron uptake and reducingiron sequestration and export, NO−dependent IRP1 activation served to maintainadequate levels of intracellular iron in order to fuel the Fe−S biosynthetic pathway, asdemonstrated by the efficient restoration of the mitochondrial Fe−S aconitase, which wasprevented under IRP1 deficiency. Furthermore, activated IRP1 was potent enough todown-regulate the abnormally increased L-Ft and H-Ft protein levels in Irp2-/-macrophages. Endogenous NO activated IRP1 IRE-binding activity and tended todecrease IRP2 IRE-binding activity. Nevertheless, IRP1 was the predominant regulator offerritin in those conditions. In hypoxia, in Irp1+/+ and Irp2+/+ macrophages exposed to NO,both stabilized IRP2 and NO-activated IRP1 seemed to cooperate to inhibit ferritinsynthesis. However, in Irp1-/- cells, IRP2 stabilized in hypoxia was sufficient to inhibit LandH-Ft synthesis despite the concomitant increase of corresponding mRNAs.Interestingly, TfR1 was shown to be predominantly regulated at the transcriptional level byNO in hypoxia, in which HIF-1 alpha may be the critical regulator. In conclusion, we revealin this study how the IRP regulon participates in the regulation of cellular iron metabolismin response to NO and its intimate interplay with the oxygen pathway. The findingsunderlie the importance to further explore the role of IRP1 in inflammation in vivo, in nonhypoxictissue microenvironments.

Pharmacological ascorbate, intravenous administration of high-dose vitamin C aimed at peak plasma concentrations ~ 20 mM, has recently re-emerged, after a controversial history, as a potential anti-cancer agent in combination with standard-of-care radiation and chemotherapy-based regimens. The anti-cancer effects of ascorbate are hypothesized to involve the auto-oxidation or metal-catalyzed oxidation of ascorbate to generate H2O2, and preclinical in vitro and in vivo studies in a variety of disease sites demonstrate the efficacy of adjuvant ascorbate. Furthermore, phase I clinical trials in pancreatic and ovarian cancer have demonstrated safety and tolerability in combination with chemotherapy and preliminary results suggest therapeutic efficacy. Both preclinical in vitro and in vivo studies as well as phase I clinical trials suggest a cell-intrinsic mechanism of selective toxicity of cancer cells as compared to normal cells; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study aims to investigate the preclinical therapeutic efficacy of pharmacological ascorbate in combination with standard cancer therapies in three novel disease sites: non-small cell lung cancer (NSCLC), glioblastoma multiforme (GBM), and some histological subtypes of sarcoma. In vitro experiments demonstrate cancer cell-selective susceptibility to pharmacological ascorbate as compared to normal cells of identical cell lineages. Furthermore, in vivo murine xenograft models of NSCLC, GBM, and fibrosarcoma demonstrate therapeutic efficacy of pharmacological ascorbate in combination with chemotherapy and/or radiation as compared to chemotherapy and/or radiation alone without any additional therapeutic toxicity. Additionally, a phase I clinical trial in GBM subjects demonstrates the safety and tolerability of ascorbate in combination with radiation and temozolomide therapy. Although not powered for efficacy, preliminary results suggest that ascorbate may be efficacious in these subjects (median survival 18.2 months vs. 14.6 months in historical controls), and, importantly, that ascorbate therapy may be independent of MGMT promoter methylation status (median survival 23.0 months vs. 12.7 months in historical controls with absent MGMT promoter methylation). Preliminary results from a phase II clinical trial of ascorbate in combination with carboplatin/paclitaxel chemotherapy in advanced stage NSCLC subjects also demonstrate promising preliminary results related to efficacy (objective response rate (ORR) 29% and disease control rate (DCR) 93% vs. historical control ORR 15-19% and DCR 40%). In addition to demonstrating the potential efficacy of pharmacological ascorbate in combination with standard anti-cancer therapies, this work demonstrates that the selective toxicity of ascorbate may be mediated by perturbations in cancer cell oxidative metabolism. Increased mitochondrial-derived O2- and H2O2 disrupts cellular iron metabolism, resulting in increased iron uptake via Transferrin Receptor and a larger intracellular labile iron pool. The larger pool of labile iron in cancer cells underlies the selective sensitivity of cancer cells to ascorbate toxicity through pro-oxidant chemistry with ascorbate-produced H2O2. This mechanism is further supported by the finding of increased levels of O2- and labile iron in patient lobectomy-derived NSCLC tissue as compared to adjacent normal fresh frozen tissue. Together, these studies demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in NSCLC, GBM, and sarcoma therapy and propose that further investigations of tumor and systemic iron metabolism are required to determine if these alterations can be exploited to enhance therapeutic efficacy or serve as therapeutic biomarkers.

[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.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Oesophageal cancer (OC) is the sixth leading cause of cancer related deaths in the world with approximately 300 000 new cases reported each year. OC may be characterized into two forms with 90% of cases presenting as squamous-cell carcinoma (SCC) and the remaining 10% as adenocarcinoma (ADC). Several factors have been attributed to the development of OC, including oesphageal injury and/or irritation, chronic inflammation and excess iron associated with enhanced tumour growth. The HAMP gene codes for a 25 amino-acid protein found to be primarily expressed in the liver and crucial to regulation of bodily iron status. Defects occurring in the HAMP gene could therefore lead to the dysregulation of the gene, resulting in an iron overload status. Iron overload is a previously described risk factor in the development of various cancers, including OC, and therefore the aim of this study was to investigate whether dysregulation of the HAMP gene may be involved in the cancer phenotype exhibition. The study cohort comprised of 48 unrelated patients presenting with SCC and a control group of 51 healthy, unrelated population-matched individuals. Mutation detection techniques included polymerase chain reaction (PCR) amplification, heteroduplex single-stranded conformation polymorphism (HEX-SSCP) analysis and bi-directional semi-automated DNA sequencing analysis. Screening of the 5’ regulatory region (5’UTR) of the HAMP gene revealed one known (-582A/G) and two novel (-188C/T and -429G/T) variants with the -429G/T variant showing statistically significant reduction in expression in patients relative to controls. Iron parameters were correlated between patient and control cohorts, as well as for variant presence and absence within individuals. Luciferase reporter constructs were used to investigate the functional implications of the presence of a variant on HAMP gene expression, and how these results correlated to the iron parameter statistics obtained. Luciferase reporter assay results indicated the -188C/T and -429G/T variants to result in under-, and the -582A/G variant to result in over-expression at the basal level, relative to the respective wild-type sequence constructs. Correlation of the luciferase data with the iron parameter statistics, indicate the -429G/T variant to be coupled to significantly higher levels of ferritin and C-reactive protein (CRP) and significantly lower levels of serum-iron and transferrin when compared to individuals without the variant. Considering only the patient group, the presence of the -188C/T and -429G/T variants were coupled to significantly lower levels of transferrin in patients with either variant, compared to patients without. The variants found within the HAMP promoter region are therefore able to alter gene regulation to an extent where iron parameters deviate between healthy and OC afflicted individuals, and also between patients with and without a variant. This dysregulation in iron homeostasis may play a role in the development and/ or progression of OC. Characterisation of the 5’ UTR of the HAMP gene may contribute to linking iron regulation to the establishment of an effective screening program, facilitating the early detection of OC. / AFRIKAANSE OPSOMMING: Slukdermkanker (SK) is die sesde grootste oorsaak van kanker-verwante sterftes in die wêreld, met sowat 300 000 nuwe gevalle wat aangemeld word elke jaar. SK kan geklassifiseer word in twee vorme, waar 90% van die gevalle plaveisel-selkarsinoom (SSC) vorm en die oorblywende 10%, adenokarsinoom (ADC). Verskeie faktore word toegeskryf aan die ontwikkeling van SK, insluitend slukderm beserings en/ of irritasie, chroniese inflammasie en oormatige ystervlakke wat geassosieer word met verhoogde gewasgroei. Die HAMP geen kodeer vir 'n 25 aminosuur proteïen wat hoofsaaklik in die lewer uitgedruk word en noodsaaklik is vir die regulering van ystervlakke in die liggaam. Defekte wat in die HAMP geen voorkom kan dus die onreëlmatige regulering van die geen tot gevolg hê, wat lei tot yster-oorlading. Yster-oorlading is voorheen beskryf as ‘n risiko faktor in die ontwikkeling van verskillende vorme van kanker, insluitend SK en gevolglik was die doel van hierdie studie om te bepaal of die wanregulering van die HAMP geen betrokke mag wees by die uitdrukking van die kanker fenotipe. Die studiepopulasie het bestaan uit 48 onverwante pasiënte met SSC en ‘n kontrole-groep van 51 gesonde, onverwante soortgelyke individue. Die mutasie opsporingstegnieke wat gebruik is, het polimerase kettingreaksie (PKR) amplifisering, heterodupleks enkelstring-konformasie polimorfisme (HEX-SSCP) analise en bidireksionele semi-outomatiese DNS volgordebepaling-analise van die geïdentifiseerde variante ingesluit. Sifting van die 5’ regulerende area (5'UTR) van die HAMP geen het een bekende (-582A/G) en twee nuwe (-188C/T en -429G/T) variante opgelewer, met die -429G/T variant wat statisties beduidend onderdruk is in pasiënt uitdrukkings vlakke relatief tot 'n gesonde kontole-groep. Yster-parameters van alle pasiënt en kontole individue is gekorreleerd tussen pasiënt en kontrole groepe, sowel as vir teenwoordigheid of afwesigheid van variante in elke individu. Luciferase verklikker konstrukte is gebruik om die funksionele implikasies van die teenwoordigheid van ‘n variant op HAMP geenuitdrukking te ondersoek, en hierdie resultate te korreleer met yster-parameter statistieke wat verkry is. Luciferase verklikkertoetse dui aan dat die -188C/T en -429G/T variante tot verminderde, en die -582A/G variant lei tot die verhoogte uitdrukking op die basale vlak lei, relatief tot die onderskeie wilde-tipe konstukte. Korrelasie van die luciferase data met die yster-parameter statistieke, dui aan dat die -429G/T-variant gekoppel is aan aansienlik hoër vlakke van feritien en C-reaktiewe proteïen (CRP) en beduidend laer vlakke van serum-yster en transferrien in vergelyking is met individue sonder die variant. Met oorweging van slegs die pasiënt-groep, is die teenwoordigheid van die -188C/T en -429G/T variante beduidend gekoppel aan laer vlakke van transferrien in pasiënte met die variant, in vergelyking met pasiënte daarsonder. Variante binne die HAMP promotor is dus in staat om geenregulasie te verander tot so 'n mate dat die yster-parameters afwyk tussen gesonde en SK geaffekteerde individue, sowel as tussen pasiënte met en sonder ’n variant. Hierdie wanregulering in yster homeostase kan 'n rol speel in die ontwikkeling en/ of die progressie van SK. Karakterisering van die 5’ regulerende area van die HAMP geen kan grootliks bydra om ysterregulasie te verbind met die implementering van ‘n effektiewe siftingsprogram, en sodoende die vroeë opsporing van SK fasiliteer.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Iron metabolism disorders comprise the most common disorders in humans. Hereditary haemochromatosis (HH) is a common condition resulting from inappropriate iron absorption. The most common form of the disease (Type 1) is associated with mutations in the HFE gene. The C282Y homozygous genotype accounts for approximately 80% of all reported cases of HH within the Caucasian population. A second HFE mutation, H63D, is associated with less severe disease expression. The C282Y mutation is extremely rare in Asian and African populations. The H63D mutation is more prevalent and has been observed in almost all populations. Iron overload resulting from haemochromatosis is predicted to be rare in Asian Indian populations and is not associated with common HFE mutations that are responsible for HH in the Caucasian population. The aberrant genes associated with HH in India have not yet been identified. The present study attempted to identify variants in six iron regulatory genes that were resulting in the Type 1 HH phenotype observed in two Asian Indian probands from a highly consanguineous family. The promoter and coding regions of the HMOX1, HFE, HAMP, SLC40A1, CYBRD1 and HJV genes were subjected to mutation analysis. Gene fragments were amplified employing the polymerase chain reaction (PCR) and subsequently subjected to heteroduplex single-strand conformational polymorphism (HEX-SSCP) analysis. Samples displaying aberrations were then analysed using bi-directional semi-automated DNA sequencing analysis to identify any known or novel variants within the six genes. Variants disrupting restriction enzyme recognition sites were genotyped employing restriction fragment length polymorphism (RFLP) analysis. Mutation analysis of the six genes revealed 24 previously identified variants, five novel variants (HFE: 5’UTR-840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR- 1272T→C; HJV: 5’UTR-534G→T, 5’UTR-530G→T), one previously described microsatellite and two novel repeats. Variants identified within the SLC40A1, CYBRD1 and HJV genes do not seem to be associated with the iron overload phenotype. A previously described HAMP variant (5’UTR-335G→T) was observed in the homozygous state in both probands. This variant seems to be the genetic aberration responsible for iron overload in this Indian family. The severe juvenile haemochromatosis phenotype usually associated with HAMP mutations, was not exhibited by the two Indian probands. Their symptoms resembled those observed in classic Type 1 HH. It is suggested that variants identified in the HMOX1 and HFE genes are modifying the effect of the HAMP variant and resulting in the less severe disease phenotype. Although this variant has only been identified in one Indian family, it could shed some light in the hunt for the iron-loading gene in India. / AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is ‘n algemene siektetoestand wat ontstaan as gevolg van oneffektiewe opname van yster in die liggaam. Die mees algemene vorm van die siekte (Tipe 1) word geassosieer met mutasies in die HFE-geen. Die C282Y homosigotiese genotipe is verantwoordelik vir ongeveer 80% van alle gerapporteerde gevalle van OH binne die Kaukasiese bevolking. ‘n Tweede HFE mutasie, H63D, word geassosieer met minder ernstige siekte simptome. Die C282Y mutasie is besonder skaars in Asiese en Afrika bevolkings. Daar word bespiegel dat oorerflike ysteroorlading as gevolg van hemochromatose skaars is in Asiese Indiër bevolkings en word nie geassosieer met algemene HFE mutasies wat verantwoordelik is vir OH in Kaukasiese bevolkings nie. Die abnormale gene wat wél geassosieer word met OH in Indië is tot dusver nog nie identifiseer nie. Die doel van hierdie studie was om die variante in ses yster-regulerende gene te identifiseer wat die Tipe 1 OH fenotipe in hierdie familie veroorsaak. Hierdie fenotipe is waargeneem in twee Asies Indiese familielede afkomstig van ‘n bloedverwante familie. Die promotor en koderingsareas van die HMOX1, HFE, HAMP, SLC40A1, CYBRD1 en HJV gene is gesif vir mutasies. Geen fragmente is geamplifiseer met behulp van die polimerase kettingsreaksie (PKR) en daarna aan heterodupleks enkelstring konformasie polimorfisme (HEX-SSCP) analise blootgestel. PKR produkte wat variasies getoon het, is daarna geanaliseer deur tweerigting semi-geoutomatiseerde DNS volgorde-bepalingsanalise om enige bekende of nuwe variante binne die ses gene te identifiseer. Variante waar restriksie ensiem herkenningsetels teenwoordig is, is verder analiseer met behulp van die restriksie fragment lengte polimorfisme (RFLP) analise sisteem. Mutasie analise van die ses gene het 24 bekende variante, vyf nuwe variante (HFE: 5’UTR- 840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR-1272T→C, HJV: 5’UTR-534G→T, 5’UTR-530G→T), een bekende herhaling en twee nuwe herhalings gewys. Variante wat binne die SLC4041, CYBRD1 en HJV gene geïdentifiseer is, blyk nie om by te dra tot die ysteroorladings-fenotipe nie. Die bekende HAMP variant (5’UTR-335G→T) is waargeneem in die homosigotiese toestand in beide van die aangetaste individue. Hierdie variant blyk om die genetiese fout te wees wat verantwoordelik is vir die ysteroorlading in die betrokke Indiese familie. Die erge juvenielehemochromatose fenotipe wat meestal geassosieer word met HAMP-mutasies, is nie waargeneem in hierdie familie nie. Hul simptome kom ooreen met die simptome van die klassieke Tipe 1 OH. Dit blyk moontlik te wees dat die variante identifiseer in die HMOX1 en HFE gene die impak van die HAMP variant modifiseer en die matiger siekte-fenotipe tot gevolg het. Alhoewel hierdie variant slegs in een Indiese familie geïdentifiseer is, kan dit lig werp op die soektog na die veroorsakende ysterladingsgeen in Indië.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007. / Hereditary haemochromatosis (HH), a common autosomal recessive disease, is characterized by increased iron absorption leading to progressive iron accumulation in organs such as the liver, heart and pancreas. In the South African population the disease is prevalent in individuals of Caucasian origin, with a carrier frequency of one in six for the C282Y mutation in the HFE gene. We investigated the role of genes implicated in iron metabolism, including the high-iron gene (HFE), haem oxgenase-1 gene (HMOX1), solute carrier family 40 (iron-regulated transporter) member 1 gene (SLC40A1), cytochrome b reductase gene (CYBRD1), hepcidin antimicrobial peptide gene (HAMP) and the hemojuvelin gene (HJV) in a patient cohort with non-HFE iron overload. DNA analysis was performed on samples from 36 unrelated South African Caucasian patients presenting with primary iron overload, who tested either negative or heterozygous for C282Y. In this study, mutation screening was performed by PCR amplification and HEX-SSCP analysis. Sixteen previously described and two novel variants were identified by semi-automated DNA sequencing. Common variants identified in the HFE gene included C282Y, H63D, IVS2+4T→C, IVS4-44T→C, IVS4+48G→A and IVS5-47G→A. The Q127H mutation in exon 3 of the HFE gene was identified in one patient, who tested negative for both C282Y and H63D. Mutation S65C was identified only in the population-matched controls and was absent in the patient group. Other previously described polymorphisms identified included the IVS5+51delTGGCTGTCTGACT deletion in HMOX1, I109 and V221 in SLC40A1, IVS1-4C→G, IVS2+8T→C and S266N, in the CYBRD1 gene and, S264 and A310G in the HJV gene. The novel variants, -89C→T, in the promoter region of the CYBRD1 gene, was detected in only one patient, while S333 in exon 4 of the HJV gene was present in three patients. These variants were not identified in any of the population-matched controls screened and could explain the non-HFE iron overload presented by these patients. This study clearly demonstrates the importance of modifier genes in patients with iron overload that cannot be explained by the common C282Y mutation. Studies on iron-related genes and the identification of mutations in these genes in non-HFE patients could lead to improved diagnosis and counselling of South African patients presenting with primary iron overload.