京都大学 / 新制・課程博士 / 博士(医学) / 甲第24134号 / 医博第4874号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 中川 一路, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Leung, Yeung Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-154). / Abstracts in English and Chinese. / CONTENTS --- p.i / ACKNOWLEDGEMENTS --- p.iv / ABSTRACT --- p.v / 論文摘要 --- p.viii / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xv / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Brain iron function and diseases --- p.1 / Chapter 1.1.1 --- Function of iron in the brain --- p.1 / Chapter 1.1.2 --- Iron involved oxidative damage --- p.2 / Chapter 1.1.3 --- Role of iron in neurodegenerative diseases --- p.6 / Chapter 1.1.4 --- Role of iron in Alzheimer's disease --- p.7 / Chapter 1.1.5 --- Deleterious effects of iron in memory function --- p.9 / Chapter 1.2 --- Iron regulation in the brain --- p.10 / Chapter 1.2.1 --- Transport and storage of brain iron --- p.10 / Chapter 1.2.2 --- Iron homeostasis in the brain --- p.14 / Chapter 1.2.3 --- Transport of iron in axon and synapse --- p.17 / Chapter 1.3 --- The hippocampus --- p.19 / Chapter 1.3.1 --- Hippocampus and memory function --- p.19 / Chapter 1.3.2 --- Structure of the hippocampus --- p.20 / Chapter 1.3.3 --- Cell composition in the hippocampus --- p.26 / Chapter 1.3.4 --- Wiring in the hippocampus --- p.28 / Chapter 1.4 --- Synaptic plasticity and long term potentiation --- p.30 / Chapter 1.4.1 --- Basic theory of synaptic plasticity --- p.30 / Chapter 1.4.2 --- Types of synaptic plasticity --- p.30 / Chapter 1.4.3 --- The discovery of long term potentiation --- p.31 / Chapter 1.4.4 --- Long term potentiation --- p.32 / Chapter 1.4.5 --- Cellular mechanism of long term potentiation --- p.33 / Chapter 1.4.6 --- Role of reactive oxygen species in long term potentiation --- p.36 / Chapter 1.5 --- Aim of the study --- p.38 / Chapter 2. --- MATERIALS AND METHODS --- p.39 / Chapter 2.1 --- Rat model of iron overload --- p.39 / Chapter 2.2 --- Multi-electrode field potential measurement --- p.40 / Chapter 2.2.1 --- Acute preparation of hippocampal slices --- p.40 / Chapter 2.2.2 --- Multi-electrode array recording system --- p.41 / Chapter 2.2.3 --- Recording of field excitatory postsynaptic potentials --- p.42 / Chapter 2.2.4 --- Induction of LTP --- p.47 / Chapter 2.2.5 --- Recording of paired-pulse ratio --- p.48 / Chapter 2.3 --- Whole cell patch-clamp recordings --- p.50 / Chapter 2.4 --- Biochemical assays --- p.57 / Chapter 2.4.1 --- Preparation of brain homogenate --- p.57 / Chapter 2.4.2 --- Total iron measurement --- p.57 / Chapter 2.4.3 --- Protein carbonyl measurement --- p.58 / Chapter 2.4.4 --- Determination of reactive oxygen species --- p.60 / Chapter 2.5 --- Drugs and data analysis --- p.61 / Chapter 3. --- RESULTS --- p.62 / Chapter 3.1 --- The acute effects of extracellular iron on synaptic transmission and long-term synaptic plasticity in the hippocampus in vitro --- p.63 / Chapter 3.1.1 --- Effects of ferric ion on basal synaptic transmission --- p.63 / Chapter 3.1.1.1 --- Effect of FAC on basal fEPSPs --- p.63 / Chapter 3.1.1.2 --- Comparison with the effect of AC on basal fEPSPs --- p.69 / Chapter 3.1.2 --- Effects of ferric ion on long-term synaptic plasticity --- p.72 / Chapter 3.1.2.1 --- Effect of acute FAC treatment on LTP --- p.72 / Chapter 3.1.2.2 --- Comparison with the effect of AC on LTP --- p.75 / Chapter 3.1.3 --- Effects of ferric chloride --- p.78 / Chapter 3.1.4 --- Effects of ascorbic acid on the action of FAC --- p.81 / Chapter 3.2 --- "The acute, in vitro effect of extracellular iron on the membrane properties and excitability of hippocampal CA1 neurons" --- p.86 / Chapter 3.2.1 --- Membrane input resistance --- p.86 / Chapter 3.2.2 --- Voltage-Current relationship --- p.88 / Chapter 3.2.3 --- Membrane excitability --- p.90 / Chapter 3.2.3.1 --- Threshold current --- p.90 / Chapter 3.2.3.2 --- Action potential firing frequency --- p.92 / Chapter 3.2.4 --- Action potential characteristics --- p.95 / Chapter 3.2.4.1 --- "Action potential amplitude, area and width" --- p.95 / Chapter 3.2.4.2 --- Rise and decay kinetics of action potential --- p.98 / Chapter 3.3 --- The chronic effects of iron-loading in the brain on hippocampal long-term synaptic plasticity --- p.100 / Chapter 3.3.1 --- Validation of the iron-overload model --- p.100 / Chapter 3.3.1.1 --- Short-term (1 week) treatment --- p.100 / Chapter 3.3.1.2 --- Long-term (4 weeks) treatment --- p.103 / Chapter 3.3.2 --- Effects of chornic iron-overloading on LTP --- p.105 / Chapter 3.3.2.1 --- Short term iron treatment --- p.105 / Chapter 3.3.2.2 --- Long term iron treatment --- p.108 / Chapter 3.3.3 --- Oxidative stress measurement --- p.111 / Chapter 3.3.3.1 --- Protein oxidation --- p.111 / Chapter 3.3.3.2 --- Reactive oxidative species level --- p.116 / Chapter 4. --- DISCUSSION --- p.120 / Chapter 4.1 --- "Acute, in vitro effects" --- p.121 / Chapter 4.2 --- "Chronic, in vivo effects" --- p.125 / Chapter 5. --- REFERENCES --- p.134

The soil-borne, rhizosphere-competent, filamentous fungus Trichoderma virens is a well-known biocontrol agent able to control pathogenic fungi through the production of antibiotics, the induction of systemic resistance in host plants, or by directly parasitizing the competing fungus. Competition for iron is another means by which Trichoderma can hinder competing microorganisms, and siderophores are a means by which microorganisms obtain iron. In silico analysis of the T. virens genome suggested that two genes putatively encoding extracellular siderophore-producing nonribosomal peptide synthetases (NRPSs) were present. In this study, a disruption was created in one of the genes, TvNPS6, to create a mutant unable to produce the NRPS TvNps6 (DeltaTvnps6). Previously, a mutant (DeltaTvsidD) had been generated with a disruption in the second gene (TvSIDD) encoding an NRPS thought to be involved in siderophore biosynthesis. A double mutant (DeltaDeltaTvsidDTvnps6) was generated by transformation of a DeltaTvsidD strain with a vector targeting disruption of TvNPS6. This resulted in transformants disrupted within both the putative siderophore-producing NRPSs. Thus, three mutants were available for analysis of the role of these genes in the ecology of T. virens. Transformants were confirmed by PCR and Southern blotting analysis. Phenotypic characterization of the mutants included both HPLC analysis of siderophore production, growth on agar and in liquid media, conidiation, germination in the presence of hydrogen peroxide, biocontrol against Pythium ultimum, in vitro confrontation against Rhizoctonia solani and growth with iron chelators to determine the contribution of reductive iron assimilation (RIA) compared to that of siderophores. The HPLC analysis demonstrated that T. virens Gv 29-8 (wild-type) produced a single siderophore peak when grown in an iron-depleted medium. This peak was not present in the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 mutants but was apparent with the DeltaTvsidD mutants. From the HPLC analysis, T. virens evidently produces a coprogen-type siderophore. Few differences were observed in the other phenotypic tests, though hydrogen peroxide showed some small inhibitory effects towards the DeltaTvnps6 mutants. The addition of chelators, which inhibit RIA, exerted some negative effects on all strains growing under iron-limited media, particularly the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 strains. This study demonstrated that although T. virens has two genes putatively encoding siderophore producing NRPSs, only the TvNPS6 gene was required for extracellular siderophore production. The greater sensitivity of the mutants towards the iron chelators suggests that unlike other other fungi studied, Trichoderma virens utilizes RIA, rather than siderophore production, as the primary means by which the fungus obtains iron in an iron-limited environment.

Anemia em idosos está relacionada ao aumento da morbidade e mortalidade desta população. As causas das anemias em idosos podem ser divididas em três grupos: anemia das doenças crônicas (ADC), anemia por deficiência de nutrientes, na qual se inclui a anemia por deficiência de ferro (ADF) e anemias de causas não-identificadas. A hepcidina constitui uma importante ligação entre defesa primária, inflamação e metabolismo do ferro. A hepcidina é induzida, principalmente, pela interleucina-6 (IL-6), atua como regulador negativo da absorção de ferro e é mediadora da retenção de ferro por monócitos e macrófagos durante inflamação ou infecção. Estudos recentes têm demonstrado o papel da produção desse hormônio por monócitos na homeostase do ferro, num modelo autócrino e parácrino. O presente trabalho teve por objetivo geral correlacionar os níveis de IL-6 produzidos por monócitos em cultura e a expressão de hepcidina em monócitos de indivíduos com ADC, com inflamação sem anemia, ADF e com anemias não-identificadas e, por objetivos específicos, verificar a eficiência de parâmetros hematológicos clássicos em avaliar o status férrico de idosos; comparar os níveis de IL-6 produzidos por células monocíticas em cultura, nos diferentes grupos de estudo, e relacioná-los com os parâmetros utilizados para caracterização das anemias; comparar os níveis de expressão de hepcidina em células monocíticas, nos diferentes grupos de estudo, e relacioná-los com o estado inflamatório e com os parâmetros utilizados para caracterização das anemias. Para isso, os pacientes foram avaliados através de parâmetro bioquímicos (glicemia, creatinina sérica, -glutamil transferase, proteínas totais e albumina, por método colorimétrico e proteína C-reativa, por imunoturbidimetria ultra-sensível) e hematológicos (hemograma completo, utilizando o contador de células Micros 45 ABX®, França, e extensão sangüínea corada por Leishman, ferritina sérica, por método imunoquimioluminescente, receptor de transferrina solúvel, por ensaio imunoenzimático e cálculo do índice sTfR/log ferritina). A determinação dos níveis de IL-6 foi feita por imunoensaio enzimático quantitativo em sobrenadante de cultura de monócitos e a dos níveis de expressão do RNAm da hepcidina em monócitos pela Reação em Cadeia da Polimerase em Tempo Real (RT-PCR). Os níveis séricos de ferritina estavam estatisticamente diminuídos na população com ADF, embora sem atingir os valores preconizados para diagnóstico de deficiência de ferro. Os níveis de receptor de transferrina solúvel (sTfR) e o índice sTfR/log ferritina estavam elevados em pacientes com ADF, porém, o índice não aumentou a sensibilidade da medida do receptor para pacientes idosos. Estes resultados obtidos sugerem que valores de normalidade para níveis de ferritina e índice receptor-ferritina devem ser revistos para a população idosa. Houve aumento da concentração de IL-6 em sobrenadante de cultura de monócitos no grupo Inflamação quando comparado com o grupo Anemia. Os níveis de IL-6 correlacionaram-se positivamente com os níveis da proteína C-reativa e número de leucócitos da população em estudo, porém, não houve correlação com os níveis de RNAm de hepcidina expressos por monócitos. A expressão de RNAm de hepcidina em monócitos mostrou correlação positiva com os níveis séricos de ferritina, porém, não foi diferente entre os grupos de estudo. / Anemia in elderly is associated with increased morbidity and mortality in this population. The causes of anemia in elderly can be divided into three groups: anemia of chronic diseases (ACD), anemia of nutrients deficiency, which include iron deficiency anemia (IDA) and unexplained anemias. Hepcidin is an important link between primary defense, inflammation and iron metabolism. The hepcidin is mainly induced by the Interleukin-6 (IL-6), it acts as a negative regulator of iron absorption and it is mediating of iron retention by monocytes and macrophages during inflammation or infection. Recent studies have been demonstrating the role of this hormone production by monocytes in the iron homeostasis, in autocrine and paracrine fashion. The general objective of this study was to correlate the levels of monocyte-derived IL-6 in culture and the monocyte hepcidin mRNA expression in patients with ACD, with inflammation without anemia, IDA and with unexplained anemias. The specific objectives are to verify the efficiency of classic haematological parameters in evaluating the iron status in elderly; to compare the levels of monocyte-derived IL-6 in culture, in different study groups, and to relate them with the parameters used for anemias characterization; to compare the levels of monocyte hepcidin mRNA expression, in different study groups, and to relate them with the inflammatory state and with the parameters used for anemias characterization. For that, the patients were evaluated by biochemical parameter (blood glucose, serum creatinine, -glutamyl transferase, total proteins and albumin, by colorimetric method and high-sensitivity C-reactive protein, measured by immunoturbidimetric assay) and hematological (complete blood count, using the cells accountant Micros 45 ABX®, and peripheral blood film for Leishmans staining morphology, serum ferritin level by immuno-quimioluminescent assay, soluble transferrin receptor, by Enzyme Linked Immuno Sorbent Assay (ELISA) and sTfR/log ferritin index). The determination of IL-6 levels was performed by quantitative ELISA in monocyte culture supernatants and monocyte hepcidin mRNA levels by Real-Time Polymerase Chain Reaction (RT-PCR). Although serum ferritin levels were statistically decreased in IDA population, it did not reach the values recommended for diagnosis of iron deficiency. The soluble transferrin receptor (sTfR) levels and the sTfR/log ferritin index were significantly higher in IDA group, however, the index did not increase the sensibility of the sTfR measure for elderly. These results suggest that normality values for ferritin levels and sTfR/log ferritin index should be reviewed for elderly population. There was increase of levels of monocyte-derived IL-6 in culture in Inflammation group compared with Anemia group. The IL-6 levels were positively correlated with C-reactive protein levels and leukocyte number of the patients, however, there was not correlation with monocyte hepcidin mRNA levels. The monocyte hepcidin mRNA levels showed positive correlation serum ferritin levels, however, it was not different between the study groups.

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2009. / Includes bibliographical references. / Iron availability and primary productivity in the oceans are intricately linked through photosynthesis. At the global scale we understand how iron addition induces phytoplankton blooms through meso-scale iron-addition experiments. At the atomic scale, we can describe the length and type of bonds that connect iron atoms to components of photosystem I, the most efficient light-harvesting complex in nature. Yet, we know little of how iron influences microbial diversity and distribution in the open ocean. In this study, we assess the influence of iron on the ecology of the numerically abundant marine cyanobacterium, Prochlorococcus. With its minimal genome and ubiquity in the global ocean, Prochlorococcus represents a model system in which to study the dynamics of the link between iron and primary productivity. To this end, we tested the iron physiology of two closely-related Prochlorococcus ecotypes. MED4 is adapted to high-light environments while MIT9313 lives best in low-light conditions. We determined that MIT9313 is capable of surviving at low iron concentrations that completely inhibit MED4. Furthermore, concentrations of Fe' that inhibit growth in culture are sufficient to support Prochlorococcus growth in the field, which raises questions about the species of iron available to Prochlorococcus. We then examined the molecular basis for the ability of MIT9313 to grow at lower iron concentrations than MED4 by assessing whole-genome transcription in response to changes in iron availability in the two ecotypes. / Genes that were differentially expressed fell into two categories: those that are shared by all (Prochlorococcus core genome) and those that are not (non-core genome). Only three genes shared between MED4 and MIT9313 were iron-responsive in both strains. We then tested the iron physiology of picocyanobacteria in the field and found that Synechococcus is iron-stressed in samples where Prochlorococcus is not. Finally, we propose a method to measure how iron stress in Prochlorococcus changes over natural gradients of iron in the oligotrophic ocean by quantifying transcription of the iron-stress induced gene, isiB. Taken together, our studies demonstrate that iron metabolism influences the ecology of Prochlorococcus both by contributing to its diversity and distinguishing it from other marine cyanobacteria. / by Anne Williford Thompson. / Ph.D.

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No. of bitstreams: 1 silva_mp_dr_bot.pdf: 2312700 bytes, checksum: 2943adfd4da085f54c9f7ba686299e2d (MD5) Previous issue date: 2016-06-24 / Item merged in doublecheck by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-22T17:41:47Z Item was identical to item(s): 150213, 144099 at handle(s): http://hdl.handle.net/11449/149817, http://hdl.handle.net/11449/144224 / A doença hepática gordurosa não alcoólica (DHGNA) é a alteração mais comum do metabolismo hepático, associada à obesidade, resistência à insulina e à síndrome metabólica. Compreende amplo espectro de alterações, que tem em comum a esteatose, em indivíduos que não consomem álcool. Além da sobrecarga de ingestão calórica, outros fatores como alterações do metabolismo do ferro podem estar relacionados à patogênese ou progressão da DHGNA. Analisamos no presente estudo as possíveis relações de marcadores do metabolismo do ferro com a intensidade das lesões hepáticas na DHGNA em indivíduos obesos. Foram estudados 88 indivíduos adultos, de ambos os gêneros, obesos (IMC >30), com diagnóstico histológico de DHGNA, acompanhados no ambulatório de Gastroenterologia e Hepatologia da FMB-Unesp. Os marcadores do metabolismo do ferro avaliados foram: níveis séricos de ferro, ferritina e transferrina, bem como o índice de saturação da transferrina (IST). A presença e intensidade da deposição de ferro no parênquima hepático foram investigadas pelo método histoquímico do Azul da Prússia (Perls). A intensidade da esteatose e o grau de fibrose foram avaliados nas biópsias hepáticas. Dos 88 indivíduos avaliados, 31 (35,2 %) apresentaram aumento dos níveis séricos de ferritina e 12 (13,6%) apresentaram valores de IST acima de 45 %. A avaliação histológica, das biópsias hepáticas, demonstrou a presença de fibrose em 48/88 casos (54,54%), sendo 21/88 (23,86%) de intensidade leve e 27/88 (30,68%) casos moderada/grave. A investigação da presença de hemossiderina demonstrou depósitos granulares finos no citoplasma dos hepatócitos periportais em 17/88 biópsias (19,32%). Não houve associação da presença de hemossiderina no parênquima hepático com a intensidade da fibrose. A análise de regressão logística identificou a presença de DMT2 ou glicemia de jejum > 100, dislipidemia e a presença de inflamação mista (lobular e portal) como fatores independentemente associados com a fibrose. Os resultados do presente estudo demonstraram que a presença do ferro no parênquima hepático é discreta em indivíduos obesos com DHGNA e provavelmente provavelmente não relacionada à patogênese ou progressão das lesões hepáticas que ocorrem nesta entidade. / The nonalcoholic fatty liver disease (NAFLD) is the most common modification of hepatic metabolism associated with obesity, insulin resistance and the metabolic syndrome. It comprises wide spectrum of changes, which have in common steatosis in individuals who do not consume alcohol. Besides calorie intake overload, other factors such as iron metabolism disorders may be related to the pathogenesis or progression of NAFLD. We analyzed in the present study the possible relationship of iron metabolism markers with the severity of liver injury in NAFLD in obese individuals. 88 adult subjects were studied, of both genders, obese (BMI> 30), with histological diagnosis of NAFLD, from ambulatory of gastroenterology and hepatology - FMB-Unesp. The markers of iron metabolism evaluated were: serum iron, transferrin and ferritin as well as the transferrin saturation index (STI). The presence and intensity of iron deposition in the liver parenchyma were investigated by histochemical method of Prussian blue (Perls). The intensity of steatosis and degree of fibrosis in liver biopsies were evaluated. Of the 88 individuals evaluated, 31 (35.2%) showed increased serum ferritin levels and 12 (13.6%) presented IST values above 45%. Histological evaluation of liver biopsies showed the presence of fibrosis in 48/88 cases (54.54%) and 21/88 (23.86%) mild and 27/88 (30.68%) moderate cases /serious. The investigation showed the presence of hemosiderin deposits in fine granular cytoplasm of periportal hepatocytes in 17/88 biopsies (19.32%). There was no association between the presence of hemosiderin in the liver parenchyma with the intensity of fibrosis. Logistic regression analysis identified the presence of T2DM or fasting glucose > 100, dyslipidemia and the presence of mixed inflammation (lobular and portal) as factors independently associated with fibrosis. The results of this study demonstrated that the presence of iron in the liver parenchyma is slight in obese subjects with NAFLD and probably not related to the pathogenesis or progression of liver damage occurring in this entity.

This investigation tested the hypothesis that iron, as a micronutrient, will affect proteins in Trichodesmium and therefore affect nitrogen fixation. Changes in proteins that are a result of iron enrichment were compared to naturally occuring diel changes. Alterations in the iron protein of nitrogenase were compared to nitrogen fixation rates using the acetylene reduction technique. The observed changes in proteins were compared in Trichodesmium colonies from the Caribbean Sea and the Sargasso Sea. Trichodesmium colonies were monitored for protein and iron content over a diel period on two cruises. The changes in protein and iron content in Trichodesmium colonies were variable but at times showed a cyclic diel pattern. Changes in protein bands on SDS-PAGE showed consistent changes in the banding pattern of a low molecular weight protein that responded to iron nutrition and time of day (Elardo and Rueter 1990). These changes were similar to changes m the iron protein of nitrogenase which also responded to changes associated with iron nutrition and time of day (Elardo 1991 ). Trichodesmium appear to alter certain proteins which appear as changes in banding patterns in response to environmental factors such as nutrients, temperature and light. My research shows that the pattern of modification of the iron protein of nitrogenase differs in colonies from the Caribbean Sea compared to those from the Sargasso Sea (Elardo 1991). The Caribbean Sea population in February had a clear pattern of active and inactive forms (day vs. night) of the enzyme. The Sargasso Sea population of Trichodesmium spp. had both forms of the enzyme at all times of the day during April and May when NO3 - is present in the euphotic zone due to recent mixing. These differences between the two populations may be due to different environmental conditions since the Caribbean Sea is permanently stratified, warmer and nutrient-depleted throughout the year. The Sargasso Sea undergoes seasonal breakdown of the thermocline during winter months, resulting in an injection of nitrate from deeper water, and minimum temperatures of 18oC.

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

[Truncated abstract] Iron is essential to most biological systems. Under pathological conditions affecting the iron metabolic pathway, iron can be deposited in the tissue in various forms. The work presented in this thesis has exploited the relationship between the magnetic and the chemical properties of tissue iron deposits to further understanding of two major pathologies, the haemoglobinopathies termed thalassaemias and the malaria parasite Plasmodium falciparum, both amongst the most common health concerns in tropical countries. The iron-specific magnetic susceptibilities ¿Fe for spleen tissue samples from 7 transfusion dependent ß-thalassaemia (ß-thal) patients and 11 non-transfusion dependent ß-thalassaemia/Haemoglobin E (ß/E) patients were measured at 37°C. Both groups of patients were iron loaded with no significant difference in the distribution of spleen iron concentrations between the two groups. There was a significant difference between the mean ¿Fe of the spleen tissue from each group. The ß/E patients had a higher mean (± standard deviation) spleen ¿Fe (1.55 ± 0.23 × 10-6 m3.kgFe -1) than the ß-thal patients (1.16 ± 0.25 × 10-6 m3.kgFe -1). Correlations were observed between ¿Fe of the spleen tissue and the fraction of magnetic hyperfine split sextet in the 57Fe Mössbauer spectra of the tissues at 78 K (Spearman rank order correlation ¿ = -0.54, p = 0.03) and between ¿Fe of the spleen tissue and the fraction of doublet in the spectra at 5 K (¿ = 0.58, p = 0.02) indicating that ¿Fe of the spleen tissue is related to the chemical speciation of the iron 2 deposits in the tissue. The biological variability of the iron-specific magnetic susceptibility of the tissue iron examined would contribute a random uncertainty of 19% to magnetic susceptibility based non-invasive measurements of tissue iron concentration. ... Magnetic susceptibility measurements were also performed on malaria parasitised red blood cells. In vitro cultures of P. falciparum were magnetically enriched up to 61-fold using high field gradient magnetic separation columns, and the magnetic susceptibility of cell contents was directly measured. Forms of haem iron were quantified spectroscopically. Further fractionations were performed such that, by controlling the fluid velocity through the column, cells with more than a critical amount of paramagnetic 3 iron were preferentially extracted. A chloroquine-sensitive (CQS) laboratory strain of parasites converted approximately 60% of host cell haem iron to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. The volumetric magnetic susceptibility of the magnetically enriched cells was found to be 0.15 ± 0.03 × 10-7 relative to the suspension medium, accounting for the enrichment of mature parasites. Comparisons of fractionation samples of two pairs of CQS and chloroquine resistant (CQR) strains showed enrichment of mature parasites was significantly greater in the CQS than the CQR strains. The results suggest the possibility of using magnetic separation columns in identifying CQR strains of P. falciparum, potentially in a diagnostic or research setting. The study also underlines the need to identify and quantify the forms of iron in CQR and CQS parasite strains as the fate of haem iron will have implications in understanding the mechanisms of chloroquine resistance.