The molecular biology of human genetic disorders is under intensive investigation at present. In those cases where the disorder is clearly defined in terms of altered gene structure, possibilities may exist for the correction of the disorder by insertion of normal genes through the process of DNA transfection. A possible method for the transfer of genetic material is by attempting to attach DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. By this means one might be able to get DNA into cells. This thesis deals with experimental work on the chemical modification of human serum transferrin by means of water-soluble carbodiimides. The resulting N-acylurea transferrins bind DNA in a reversible manner. Characteristics and properties of the binding interactions are dealt with in detail. N-acylurea derivatives of transferrin were prepared with the water-soluble carbodiimides, N-ethyl-N' -(3-dimethylaminopropyl) carbodiimide and N-ethyl-N' -(3-trimethylpropylammonium) carbodiimide iodide. Reactions were carried out under mild conditions at room temperature for 48-72 hours. [³ H] N-ethyl-N' -(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the protein. Changes in charge properties were determined by agarose gel electrophoresis. Carbodiimide modification of proteins is thought to occur at side chain carboxyl groups of glutamic and aspartic acid residues. This was confirmed by the use of Staphylococcus aureus V8 protease, which cleaves peptide bonds at the carboxyl side of glutamic and aspartic acid residues, but not in the case of substituted side chain carboxyl groups. Through the use of puromycin as a nucleophile it has been shown that other functional groups were not activated upon reaction of transferrin with carbodiimide. The carbodiimide-modified proteins bind various types of DNA and RNA in a reversible manner. Low concentrations of N-acylurea transferrin retarded the migration of pBR322 DNA, M 13 mp 8 single-stranded DNA and Pst 1 restricted lambda DNA on agarose gel electrophoresis, while at higher concentrations the DNA was unable to enter the gel. Nitrocellulose filter binding assays showed that binding of DNA to Nacylurea transferrins was rapid, dependent on concentration of the modified transferrin and sensitive to ionic conditions. Binding was found to occur mainly through electrostatic interactions between phosphate groups of DNA and N-acylurea groups. These conclusions were based on experiments which showed that protein-DNA complexes were dissociated by increasing salt concentrations and by heparin. Non-electrostatic interactions such as hydrophobic interactions and hydrogen bonding are also involved in binding, since half dissociation of complexes, induced by chaotropic salts, KSCN and NaC10₄occurs at lower concentrations of salt than in the case of NaCl. Also RNA polynucleotides inhibit binding of DNA to Nacylurea transferrins to varying extents. The N-acyl urea transferrins have been shown to bind certain specific restriction endonuclease cleavage sites on pBR322 DNA. The N-acylurea transferrin-DNA complexes would thus be suitable for experiments in cell transfections using cells which have transferrin receptors. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

Thesis (Ph.D.)--University of Western Australia, 2004.

Thesis (PhD)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: John W. Peters. Includes bibliographical references (leaves 131-147).

In der vorliegenden Dissertation wurden Neurone untersucht, die von einer speziellen Form der extrazellulären Matrix, dem perineuronalen Netz (PN), umgeben sind. Neurone mit einem PN zeichnen sich durch eine geringe Vulnerabilität bei neurodegenerativen Erkrankungen aus. Da das PN mit hoher Affinität Eisen bindet, war zu klären, ob das PN den Eisenhaushalt der Neurone beeinflusst und diese mit einer protektiven Eigenschaft gegenüber Eisen-induzierten oxidativen Stress ausstattet. Es wurde die Eisenkonzentration und der Gehalt an Eisentransport- und Eisenspeicherproteinen von Neuronen mit PN und Neuronen ohne PN in der Ratte untersucht. Dabei kamen quantitative Methoden wie die ortsaufgelöste Ionenstrahlmikroskopie und die Objektträger-basierte Laser Scanning Zytometrie sowie Western Blot Analysen und quantitative Real-Time-PCR zum Einsatz. Die Untersuchungen zeigen, dass Neurone, die mit einem PN umgeben sind, eine höhere Konzentration an Eisen sowie Eisentransport- und Eisenspeicherproteinen besitzen als Neurone ohne ein PN. Das PN könnte so den Eisenhaushalt der Neurone beeinflussen und diese mit einer protektiven Eigenschaft gegenüber Eisen-induziertem oxidativen Stress ausstatten.

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 .

[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.

In der vorliegenden Dissertation wurden Neurone untersucht, die von einer speziellen Form der extrazellulären Matrix, dem perineuronalen Netz (PN), umgeben sind. Neurone mit einem PN zeichnen sich durch eine geringe Vulnerabilität bei neurodegenerativen Erkrankungen aus. Da das PN mit hoher Affinität Eisen bindet, war zu klären, ob das PN den Eisenhaushalt der Neurone beeinflusst und diese mit einer protektiven Eigenschaft gegenüber Eisen-induzierten oxidativen Stress ausstattet. Es wurde die Eisenkonzentration und der Gehalt an Eisentransport- und Eisenspeicherproteinen von Neuronen mit PN und Neuronen ohne PN in der Ratte untersucht. Dabei kamen quantitative Methoden wie die ortsaufgelöste Ionenstrahlmikroskopie und die Objektträger-basierte Laser Scanning Zytometrie sowie Western Blot Analysen und quantitative Real-Time-PCR zum Einsatz. Die Untersuchungen zeigen, dass Neurone, die mit einem PN umgeben sind, eine höhere Konzentration an Eisen sowie Eisentransport- und Eisenspeicherproteinen besitzen als Neurone ohne ein PN. Das PN könnte so den Eisenhaushalt der Neurone beeinflussen und diese mit einer protektiven Eigenschaft gegenüber Eisen-induziertem oxidativen Stress ausstatten.