Iron is essential for virtually all organisms, yet it can be highly toxic if not properly regulated. Only the Lyme disease pathogen Borrelia burgdorferi has evolved to not require iron (Aguirre et al., 2013).Recent findings have characterised elements of the iron metabolism network, but understanding of systemic iron regulation remains poor. To improve understanding and provide a tool for in silico experimentation, a computational model of human iron metabolism has been constructed. COPASI was utilised to construct a model that included detailed modelling of iron metabolism in liver and intestinal cells. Inter-cellular interactions and dietary iron absorption were included to create a systemic computational model. Parameterisation was performed using a wide variety of literature data. Validation of the model was performed using published experimental and clinical findings, and the model was found to recreate quantitatively and accurately many results. Analysis of sensitivities in the model showed that, despite enterocytes being the only route of iron uptake, almost all control over the system is provided by reactions in the liver. Metabolic control analysis identified key regulatory factors and potential therapeutic targets. A virtual haemochromatosis patient was created and compared to a simulation of a healthy human. The redistribution of control in haemochromatosis was analysed in order to improve our understanding of the condition and identify promising therapeutic targets. Cellular prion protein (PrP) is an enigmatic protein, implicated in disease when misfolded, but its physiological role remains a mystery. PrP was recently found to have ferric-reductase capacity. Potential sites of ferric reduction were simulated and the findings compared to PrP knockout mice experiments. I propose that the physiological role of PrP is in the chemical reduction of endocytosed ferric iron to its ferrous form following transferrin receptor-mediated uptake.

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.

abstract: Abnormally low or high blood iron levels are common health conditions worldwide and can seriously affect an individual’s overall well-being. A low-cost point-of-care technology that measures blood iron markers with a goal of both preventing and treating iron-related disorders represents a significant advancement in medical care delivery systems. Methods: A novel assay equipped with an accurate, storable, and robust dry sensor strip, as well as a smartphone mount and (iPhone) app is used to measure total iron in human serum. The sensor strip has a vertical flow design and is based on an optimized chemical reaction. The reaction strips iron ions from blood-transport proteins, reduces Fe(III) to Fe(II), and chelates Fe(II) with ferene, with the change indicated by a blue color on the strip. The smartphone mount is robust and controls the light source of the color reading App, which is calibrated to obtain output iron concentration results. The real serum samples are then used to assess iron concentrations from the new assay and validated through intra-laboratory and inter-laboratory experiments. The intra-laboratory validation uses an optimized iron detection assay with multi-well plate spectrophotometry. The inter-laboratory validation method is performed in a commercial testing facility (LabCorp). Results: The novel assay with the dry sensor strip and smartphone mount, and App is seen to be sensitive to iron detection with a dynamic range of 50 - 300 µg/dL, sensitivity of 0.00049 µg/dL, coefficient of variation (CV) of 10.5%, and an estimated detection limit of ~15 µg/dL These analytical specifications are useful for predicting iron deficiency and overloads. The optimized reference method has a sensitivity of 0.00093 µg/dL and CV of 2.2%. The correlation of serum iron concentrations (N=20) between the optimized reference method and the novel assay renders a slope of 0.95, and a regression coefficient of 0.98, suggesting that the new assay is accurate. Lastly, a spectrophotometric study of the iron detection reaction kinetics is seen to reveal the reaction order for iron and chelating agent. Conclusion: The new assay is able to provide accurate results in intra- and inter- laboratory validations and has promising features of both mobility and low-cost. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2020

Trichomonas vaginalis is a human pathogen that affects annually approximately 258 million people worldwide. This parasite possesses organelles of mitochondrial origin called hydrogenosomes, which generate ATP under anaerobic conditions. The identification of the protein content at the subcellular level may provide new targets for antiparasitic drugs developments as well as it contributes for our understanding of the organelles function and evolution. The availability of protocols for organelles purification and the complete genome sequence allow the study of the organellar proteomes using mass spectrometry and bioinformatics, providing a powerful strategy that combine cell biology and proteomics. In our research, we used several approaches to identify the protein composition in hydrogenosomes and mitosomes. We performed transcriptomic and proteomic analysis to investigate the molecular responses of Trichomonas vaginalis upon iron availability. Furthermore, the changes in the proteome during the development of metronidazole resistance were also studied. The organelles separated by differential and Optiprep-sucrose gradient centrifugation were analyzed with nano- RP-HPLC/MALDI-TOF/TOF. We also used Triton X-114 phase partitioning to separate membrane proteins and iTRAQ technique to label the peptides...

Iron is an essential nutrient for growth of photosynthetic microorganisms such as cyanobacteria and algae. Iron is required for proteins involved in the important processes of carbon and nitrogen assimilation. Low concentrations of iron in cultures or natural waters can lead to iron limitation which affects many aspects of algal metabolism. In natural waters, iron limitation can have effects on the patterns and rates of primary productivity. The cellular content of certain proteins can be affected by media iron concentrations. Methods have been used that assay components of the cell as an indirect measure of iron nutritional status. For example, spectroscopy can be performed to determine the cellular concentration of iron-containing proteins involved in photosynthesis. Organisms grown in media that imitate natural conditions, or organisms collected from their natural habitat are usually dilute. Methods that assay iron nutritional status such as spectroscopy and column chromatography require large sample sizes which are difficult to obtain from natural samples. In addition, methods that utilize techniques such as immunology or radioactive labelling are complex and time-consuming. These considerations led to the necessity of developing a technique that would be simple, rapid and effective on dilute samples. The method developed here utilized fast protein liquid chromatography (FPLC), which fulfilled these requirements. A complete analysis could be done within two to three hours with minimal sample treatment. The FPLC was simple to operate and was effective on a sample containing less than 100 μg of protein. Some photosynthetic organisms, when iron-depleted, can produce the flavin-containing protein flavodoxin (Flv). This protein substitutes for the iron-containing protein ferredoxin (Fd) in Fd-dependent reactions such as the light-induced reduction of NADP. The FPLC technique identified and quantified, in relative terms, Fd and Flv in the cell. Optical spectroscopy was used to verify FPLC retention time assignments. The results illustrated how the FPLC could be used to observe the changes in relative Fd and Flv content as a function of media iron concentration in cultures of the cyanobacterium Anabaena grown in the laboratory. It was found that Fd content decreased and Flv content increased with decreasing media iron concentration. In addition, samples of the cyanobacterium Trichodesmium collected from the ocean near Barbados were analyzed using FPLC to assay relative Fd and Flv content. By analogy with Anabaena, Fd and Flv retention times were identified. Using this technique conclusions could be drawn regarding the changing iron nutritional status of Trichodesmium in its natural habitat .

No description available.

No description available.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Nutrition and Food Science, 1976 / Vita. / Includes bibliographical references. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Nutrition and Food Science

No description available.

No description available.