Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2004.

Background: Clinical studies have shown that tea consumption leads to decreased iron absorption. This finding is however, not supported by epidemiological studies, where no relationship between an increased tea consumption and a lower iron status in a population at risk of iron depletion has been found. Objectives: The main aim of this study was to compare the effects of black tea and Rooibos consumption on the iron status of primary school children in a rural setting in Potchefstroom, South Africa. Methods: One hundred and seventy five children, aged six to fifteen years, participated in this single blind, randomised, parallel intervention trial. Subjects were randomly allocated to receive two 200ml servings of either black tea or Rooibos with milk and sugar. These beverages were consumed during breaks and at the same time as the food h m the school-feeding scheme. The trial proceeded for sixteen weeks. The children received antihelminthic treatment (500mg mebendazole) at baseline. Haemoglobii haematocrit, serum iron, ferritin and transferfin were measured and total iron binding capacity and transferrin saturation were calculated. Trained fieldworkers measured dietary intakes by means of 24-hour dietary recalls and anthropometrists took anthropometric measurements. All the above mentioned data were gathered at the beginning and at the end of the intervention period. Results: Measurements indicated a study population that is malnourished in terms of anthropometrical indices and nutrient intakes. Biochemical markers of iron status also indicated that the population could be at risk of iron depletion. Changes in red blood cell count, haemoglobin, haematocrit, mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), serum iron, transferrin, transferrin saturation, ferritin and total iron binding capacity (TIBC) did not differ significantly between the two groups. Mean red blood cell count, haematocrit, MCV, transferrin and TIBC increased significantly h m baseline to end in both groups (all p<0.0001) and MCH decreased significantly (p<.0001). Mean haemoglobin increased significantly with black tea consumption (p=0.002), although not with the consumption of Rooibos (p=0.073). Conclusion: Black tea or Rooibos consumption has similar effects on the iron status of primary school children. Iron status was not compromised by black tea in comparison with Rooibos. This questions the proposed limitation of black tea consumption as a public health strategy in order to combat iron deficiency in a population with marginal iron status. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2004.

Background: Clinical studies have shown that tea consumption leads to decreased iron absorption. This finding is however, not supported by epidemiological studies, where no relationship between an increased tea consumption and a lower iron status in a population at risk of iron depletion has been found. Objectives: The main aim of this study was to compare the effects of black tea and Rooibos consumption on the iron status of primary school children in a rural setting in Potchefstroom, South Africa. Methods: One hundred and seventy five children, aged six to fifteen years, participated in this single blind, randomised, parallel intervention trial. Subjects were randomly allocated to receive two 200ml servings of either black tea or Rooibos with milk and sugar. These beverages were consumed during breaks and at the same time as the food h m the school-feeding scheme. The trial proceeded for sixteen weeks. The children received antihelminthic treatment (500mg mebendazole) at baseline. Haemoglobii haematocrit, serum iron, ferritin and transferfin were measured and total iron binding capacity and transferrin saturation were calculated. Trained fieldworkers measured dietary intakes by means of 24-hour dietary recalls and anthropometrists took anthropometric measurements. All the above mentioned data were gathered at the beginning and at the end of the intervention period. Results: Measurements indicated a study population that is malnourished in terms of anthropometrical indices and nutrient intakes. Biochemical markers of iron status also indicated that the population could be at risk of iron depletion. Changes in red blood cell count, haemoglobin, haematocrit, mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), serum iron, transferrin, transferrin saturation, ferritin and total iron binding capacity (TIBC) did not differ significantly between the two groups. Mean red blood cell count, haematocrit, MCV, transferrin and TIBC increased significantly h m baseline to end in both groups (all p<0.0001) and MCH decreased significantly (p<.0001). Mean haemoglobin increased significantly with black tea consumption (p=0.002), although not with the consumption of Rooibos (p=0.073). Conclusion: Black tea or Rooibos consumption has similar effects on the iron status of primary school children. Iron status was not compromised by black tea in comparison with Rooibos. This questions the proposed limitation of black tea consumption as a public health strategy in order to combat iron deficiency in a population with marginal iron status. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2004.

Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

The transferrin iron acquisition system of Neisseria consists of two dissimilar proteins, transferrin binding protein A and B (TbpA and TbpB). TbpA and TbpB both specifically and independently bind human transferrin (Tf). TbpA is a TonB-dependent transporter, expression of which is necessary for Tf iron acquisition. In contrast, the lipoprotein TbpB is not necessary for iron internalization; however it makes this process more efficient. The role of TbpB in the transferrin iron acquisition system has not been completely elucidated. It has been suggested that TbpB is entirely surface exposed and tethered to the outer membrane by its lipid moiety. We inserted the hemagluttinin antigen (HA) epitope into TbpB in an effort to examine surface accessible and functional domains of the lipoprotein. We determined that TbpB was entirely surface exposed from just beyond the mature N-terminus. It was previously reported that the N- and C-terminus of TbpB independently bind Tf. HA epitope analysis defined both the N-terminal and C-terminal binding domains. TbpB was previously reported to play an important role in the release of Tf from the receptor. We established that TbpB exhibited a biphasic dissociation pattern; a C-terminal rapid release followed by a slower N-terminal release. These results suggested that the C-terminus plays a role in ligand turnover of the wild-type receptor. Little is known about the transport of TbpB to the outer membrane. In an attempt to identify the signals/mechanisms required for TbpB localization, the signal sequence of the protein was altered. In the absence of lipid modification, TbpB remained associated with the cell, localized to the periplasm. We also noted that internal cysteine residues were not critical for TbpB localization. Our results suggested that TbpB was transported by a lipoprotein-specific mechanism. Additionally, we demonstrated the major outer membrane secretin, PilQ, was not necessary for proper localization of TbpB. The mechanism responsible for this process remains elusive. This body of work represents the first comprehensive study of TbpB topology and function, utilizing the lipoprotein expressed in its native membrane. These results may translate to other, similar lipoprotein receptors of the pathogenic Neisseria, helping to shed light on these poorly understood proteins.

Transferrin är ett protein som har uppgiften att transportera järn i kroppen. Transferrin är ett glykoprotein med två kolhydratkedjor med två, tre förgreningar med terminala sialinsyror. Antalet sialinsyror ger proteinet olika isoformer och benämns därefter, så som tetrasialotransferrin som har fyra terminala sialinsyror och är den vanligaste förekommande isoformen. Disialotransferrin används som en biokemisk markör för att identifiera individer som har en hög alkoholkonsumtion under en längre period. Disialotransferrin ökar i koncentrationen vid hög alkoholkonsumtion under minst 14 dagar. Den benämns som kolhydratfattigt transferrin (eng. Carbohydrate-Deficient Transferrin, CDT) och analyseras med jonbyteskromatografi. Referensvärdet för CDT är &gt; 2,0 % då en individ har ett alkoholmissburk. Syftet med studien var att undersöka om svarstiden går att förkorta genom att optimera upparbetningen av prover och sedan utföra en metodverifiering. Optimeringen av upparbetningen omfattade en kortare inkuberingstid från nästan ett dygn till 60 minuter, förenklad tillsättning av fällningsreagens och förändrad centrifugering av proverna. I ett inledande försök analyserades 25 serumprover med olika varianter av optimerad upparbetning och resultatet jämfördes mot standardmetoden. Metodverifieringen bestod av analys av 35 serumprover med den nya upparbetningsmetoden vilket jämfördes mot standardmetoden. I analysen jämfördes även precisionen för låg (1,4 % CDT) och hög (3,2 % CDT) kontroll samt två patientprover med ett låg och hög halt av disialotransferrin med den nya metoden. Precisionen för den låga kontrollen och patientprov 1 (CV=18,05 %) visade sig var sämre än för den höga kontrollen och det patientprov 2 (CV=5,79 %). Analys av de 35 proverna visade att det var en bra överensstämmelse mellan metoderna; korrelationskoefficienten var 0,997 och ett parat t-test kunde inte detektera någon statistik signifikant skillnad mellan proverna (95 % konfidensnivå). På grund av den sämre precisionen för låga koncentrationer av disialotransferrin behöver dock den nya upparbetningsmetoden utvärderas ytterligare. Däremot är det bestämningen av CDT-halten runt 2 % som är viktig och den nya metoden har inte sämre variationskoefficient (CV %) än standardmetoden.

O ferro é um elemento chave em muitos processos metabólicos, como transporte de oxigênio, síntese de hormônios esteróides, respiração celular, transporte de elétrons, síntese de DNA, proliferação e diferenciação celular e regulação gênica. A deficiência de ferro é a desordem nutricional mais comum afetando aproximadamente um terço da população mundial. Pequenos déficits no compartimento funcional de ferro têm sérias conseqüências sobre o sistema imune, principalmente na imunidade mediada por células. A abordagem dos pais ou responsáveis, as exigências éticas e a aderência de crianças da mesma faixa etária e sem outros problemas que afetem o metabolismo do ferro e o sistema imune são as principais dificuldades enfrentadas no desenvolvimento de pesquisas com seres humanos, sendo necessário o estabelecimento de modelos experimentais. Este trabalho teve como objetivo estabelecer um modelo de indução e recuperação de deficiência de ferro em camundongos, visando a sua utilização em estudos sobre alterações do sistema imune induzidas por esta deficiência. A deficiência de ferro foi induzida por ingestão de uma ração com baixo teor de ferro (5 mg /kg de ração) por 4 e 8 semanas. No termino deste período foram determinados: concentração de hemoglobina (colorimetrico), hematócrito (microhematócrito), estoques de ferro hepático (espectrometria de absorção atômica) e fenotipagem (citometria de fluxo) dos linfócitos presentes no sangue periférico e em suspensão de células do baço dos animais dos grupos controle (C) e deficiente em ferro (DF), sendo avaliado a porcentagem de células T CD4+ e CD8+, bem como a expressão do receptor de transferrina (CD71+) nessas subpopulações. Não houve diferenças na concentração de hemoglobina e no valor do hematócrito entre os animais dos grupos DF e C, porém os estoques de ferro estavam significantemente reduzidos nos animais do grupo DF de quatro (p<0,05) e oito (p<0,01) semanas. Não houve diferenças na porcentagem de linfócitos T CD4+ e T CD8+ entre os animais dos grupos DF e C, porém os animais deficientes em ferro apresentaram maior porcentagem de linfócitos T CD8+ do baço expressando CD71+ (p< 0,001). Este trabalho sugere que a depleção nos estoques de ferro não altera a proporção dos subtipos de linfócitos, porem as células T CD8 + do baço são mais sensíveis à deficiência de ferro. / Iron have a crucial role in several metabolic pathways, such oxygen transport, steroid hormone synthesis, cellular respiration, electron transport, DNA synthesis, cellular proliferation and differentiation and genic regulation. The iron deficiency is most common disorder nutrition, affecting about 30% world population. Deficits in iron functional compartment have serious delays about immunity systems, especially in the cellular immunity. Because of environmental problems, age, deficiency of nutrients other than iron, prevalence of infection, which may make human studies difficult, we used an animal model. This work aimed established iron deficiency induction and recuperation in mouse, for study about immune systems alteration. Iron deficiency was induced by feeding mice a diet that contained only 5 mg Fe/Kg for 4 and 8 weeks. After this period were determined: hemoglobin (colorimetry), hematocrit (microhematocrit), liver iron stores (atomic absorption spectrophotometer) and we performed a flow cytometry analyses in peripheral blood and spleen lymphocytes in control (C) and iron deficient (ID) mouse. We defined the effects of iron deficiency on T-cell subset and expression of cell-surface transferrin receptor (CD71+) in these cells. Hemoglobin concentration and hematocrit of ID mice were not difference those of C mice, but iron stores of ID mice (4 and 8 weeks) were reduced (p< 0,05 and p< 0,01; respectively). Although T-cells subsets in peripheral blood and spleen were not altered, iron deficiency significantly increased the number of spleen T CD8+ cells that express CD 71+ (p< 0,001). Data suggest that depletion of iron storage not alter T-cells subsets and spleen T CD8+ is the most sensible subset in iron deficiency.

Bovine lactoferrin (BLF) and bovine transferrin (BTF) are major-iron transport and regulation proteins found in bovine whey. BLF and BTF must interact with the eukaryotic cell surface to mediate their biological function of iron delivery and cellular functions of inflammatory and immunological modulation. As common components of the eukaryotic cell surface, gangliosides were used for affinity purification of BLF and BTF. Bovine gangliosides were isolated from fresh buttermilk and covalently immobilized onto controlled-pore glass beads (66 μg/g beads). After the matrix was loaded with whey protein (WPI or WPC), lactoferrin was eluted with 1 M NaCl and lll identified by N-terminal protein sequencing. Pretreated whey isolate (1 % wt/vol) showed the highest lactoferrin purity with 40% among protein sources, and whey protein isolate (10% wt/vol) showed the highest recovery with 105%. Bovine transferrin was eluted with sodium phosphate buffers at pH 7 after the immobilized matrix was loaded with a 2% (wt/vol) whey solution. The ganglioside column resulted in a 74.2% recovery of BTF from whey, and the BTF was enriched to 61% purity after Mono-Q chromatography. Bovine transferrin was identified by SDS-PAGE analysis, Western analysis, and isoelectrofocusing. In conclusion, immobilized gangliosides can be used to purify BLF and BTF from bovine whey.

This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology. In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis. Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro. In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells. / QC 20100729

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