Tillgängligheten av blod kan vara livsavgörande för många personer, därför är blodgivning en viktig del av vården. För blodgivare kan en regelbunden blodgivning påverka kroppens järndepåer vilket på sikt kan leda till en järnbrist. Syftet med denna studie var att kartlägga blodgivares järndepåer i Region Jönköpings län genom att (i) utvärdera antalet och andelen nyanmälningar som ej uppfyllt godkänd S-ferritinnivå för blodgivning, (ii) jämföra nivåerna av S-ferritin vid nyregistrering och vid upprepad blodgivning, (iii) utvärdera intag av järnprofylax hos blodgivare, (iv) jämföra antalet, andelen samt orsaken till kontrollprovtagningar före och efter S-ferritin infördes som provtagningsrutin. Data insamlades och erhölls från Blodcentralen, Jönköping, Sverige, och analyserades statistiskt. Resultatet visade att fler kvinnor än män ej blev godkända som blodgivare på grund av S-ferritin <22 µg/L. Genom att sänka S-ferritinnivån kan fler kvinnor godkännas för blodgivning. Vidare visade studien att upprepad blodgivning leder till minskade järndepåer. Grupperna som erbjudits järnprofylax hade stabilare S-ferritin över tid vilket antyder att alla blodgivare skulle gynnas av järnprofylax, men vidare studier krävs. Majoriteten av blodgivare med S-ferritin ≤100 µg/L intar erbjudna järntabletter vilket visade att blodgivarna möjliggjorde fortsatt blodgivning. Slutligen visade studien att sedan S-ferritin infördes som provtagningsrutin 2017 har kontrollprovtagningar på grund av lågt B-Hb minskat, vilket visar att risken för att utveckla en järnbrist på grund av upprepad blodgivning har minskat i Region Jönköpings län. / The availability of blood could be crucial for many people; blood donation is, therefore, a vital part of healthcare. Continuous blood donation could affect the blood donors’ iron depots which could lead to an iron deficiency. The aim of the present study was to evaluate and highlight iron depots amongst blood donors in Jönköping County by (i) evaluating the number and proportion of new applicants which did not meet the approved S-ferritin levels for blood donation, (ii) compare the levels of S-ferritin in new registrations and repeated blood donations, (iii) evaluate intake of iron supplements amongst blood donors, (iv) compare the number, proportion and cause for control sampling before and after S-ferritin was introduced as a sampling routine. Data was collected from the Department of Transfusion Medicine, Jönköping, Sweden, and analysed statistically.The result showed that more women than men were not approved for blood donation because of S-ferritin <22 µg/L. By lowering S-ferritin, additional women will be approved for blood donation. Further, the study showed that continuous blood donation leads to decreased iron depots. Groups that were offered iron supplements showed more stable S-ferritin levels over time which implied that all blood donors would benefit from iron supplements, but further studies are required. The majority of the blood donors with S-ferritin ≤100 µg/L were taking their offered iron supplements which showed that the blood donors enabled future blood donations. Lastly, since S-ferritin was introduced as a sampling routine in 2017 the amount of control samplings due to low B-Hb has decreased. This shows that the risk to develop an iron deficiency due to continuous blood donation has decreased in Jönköping County.

Einleitung: Bei Fohlen ist bisher wenig bekannt über die Eisenparameter im Blut. Das Spurenelement Eisen ist für Säugetiere essenziell, seine Hauptaufgabe ist der Sauerstofftransport. Adulte Pferde nehmen das benötigte Eisen mit dem Raufutter auf, welches in der Regel ausreichend hohe Eisengehalte aufweist. Das Hauptmolekül zur Eisenspeicherung ist Gewebe-Ferritin. Das im Serum vorkommende Serum-Ferritin spiegelt die Gesamteisenmenge des Körpers wider. Im Rahmen einer systemischen Entzündung sinkt der Serum-Eisenspiegel, da Eisen ins Gewebe umverteilt wird und in das Gebiet der Entzündung abwandert. Für die Parameter des Eisenstoffwechsels gibt es keine Referenzwerte für Fohlen, für adulte Pferde gibt es lediglich einen Referenzbereich für Serum-Eisen von 1,00-3,61 mg/l. Es gibt keine Studien, bei denen die Eisenparameter von Fohlen im Alter von ein bis sechs Monaten bestimmt wurden. Ziel der Studie: Ziel der vorliegenden Arbeit war es, die Parameter des Eisenstoffwechsels im Serum bei akuter systemischer Entzündung bei ein bis sechs Monate alten Fohlen zu untersuchen. Außerdem sollte überprüft werden, inwiefern diese Veränderung des Eisenstoffwechsels zu einem Abfall der Serum-Eisenwerte führt. Tiere, Material und Methoden: In dieser prospektiven Studie wurden EDTA-Vollblut- und Serumproben von 66 gesunden Fohlen und 84 Fohlen mit einer akuten systemischen Entzündung in Form einer abszedierenden Bronchopneumonie genommen. Die Fohlen waren im Median 109 Tage alt. Die Diagnose 'Bronchopneumonie' wurde mittels Ultraschalldiagnostik gestellt, die Fohlen hatten am Tag der Probennahme einen sonografisch ermittelten Abszesscore von über 15 cm sowie eine rektal gemessene Körperinnentemperatur über 39,0 °C. Die gesunden Fohlen wiesen eine unauffällige klinische Allgemeinuntersuchung sowie Lungenultraschalluntersuchung am Tag der Probennahme sowie in der vorherigen und darauffolgenden Woche auf. Im Labor wurde der Serum-Amyloid-A-Gehalt bestimmt. Die gesunden Pferde wiesen SAA-Werte innerhalb des Referenzbereiches auf, bei den kranken Pferden bestätigte ein Wert > 7µg/ml die Diagnose einer akuten systemischen Entzündung. Aus der EDTA-Vollblutprobe wurde innerhalb von einer Stunde mittels Durchflusszytometrie die Blutleukozytenzahl bestimmt. Die Analyse der Serumproben erfolgte im Labor LABOKLIN mit dem Analysesystem Cobas 8000 von Roche. Die statistische Auswertung der Daten erfolgte mit dem Statistikprogramm SPSS (IBM Statistics 27). Die Daten der Gesamtpopulation (n = 150) waren nicht normalverteilt. Es wurde ein Mann-Whitney-U-Test als nicht parametrischer Test für zwei unabhängige Stichproben durchgeführt, um signifikante Unterschiede zwischen den Gruppen zu ermitteln. Das Signifikanzniveau wurde auf p < 0,05 festgelegt. Ergebnisse: Bei der Auswertung der Probanden ergab sich für die gesunden Fohlen ein medianer SAA-Wert von 3,50 µg/ml, der der kranken Fohlen liegt mit 541 µg/ml hochsignifikant darüber. Die Bestimmung des Serum-Ferritin-Wertes ergab für die Gruppe der gesunden Fohlen einen Median von 3,80 µg/l (IQR 2,00-4,40 µg/l), der Median für die Gruppe der kranken Fohlen ist mit 4,60 µg/l (IQR 2,48-6,40 µg/l) signifikant (p = 0,003) höher. Die Serum-Eisenwerte der gesunden Fohlen lagen innerhalb des laborinternen Referenzbereichs für adulte Pferde (Median 1,57 mg/l; IQR 1,21-1,79 mg/l), auch die weiteren Bluteisenwerte entsprachen denen adulter Pferde. Die kranken Fohlen zeigten signifikant niedrigere Serum-Eisenwerte (Median 0,550 mg/l; IQR 0,368-0,778 mg/l), signifikant höhere Ferritin- und UIBC-Werte sowie eine signifikant geringere Eisensättigung. Die Auswertung der Leberenzymaktivitäten aus dem Serum ergab für die kranken Fohlen bei allen gemessenen Parametern im Durchschnitt niedrigere Werte als für die gesunden Fohlen. Bei dem Spurenelement Kupfer liegt der Median der gesunden Fohlen mit 18,8 µmol/l (IQR 16,4-20,1 µmol/l) im oberen Bereich der Referenzwerte von 7,9-21 µmol/l, der Median der kranken Fohlen liegt mit 25,7 µmol/l (IQR 23,1-29,0 µmol/l) signifikant über den Werten der gesunden Fohlen (p < 0,001). Schlussfolgerung: Durch eine akute systemische Entzündung kommt es zu einem Abfall des Serum-Eisens durch Umverteilung ins Gewebe und Abwanderung ins Entzündungsgebiet. Da es jedoch sowohl in der Literatur als auch in den Ergebnissen dieser Studie keine Hinweise auf einen absoluten Eisenmangel beim Fohlen gibt, sollte von einer Eisensupplementation in jedem Fall abgesehen werden.:1 Einleitung 1 2 Literaturübersicht 2 2.1 Vorkommen und chemische Eigenschaften von Eisen 2 2.2 Eisenmetabolismus 2 2.2.1 Absorption 2 2.2.2 Transport 3 2.2.3 Speicherung, Mobilisierung 4 2.2.4 Exkretion, Verlust 5 2.3 Physiologische Funktionen 6 2.4 Feststellung des Eisenstatus 7 2.5 Eisenbedarf des Pferdes 9 2.6 Eisengehalt von Futtermitteln 10 2.7 Eisenmangel 11 2.8 Supplementation von Eisen 12 2.9 Eisenüberschuss, Toxizität 12 2.10 Bakterieller Eisenstoffwechsel 13 2.11 Entzündung 14 2.11.1 Abszedierende Bronchopneumonie 15 2.12 Eisenstoffwechsel bei systemischer Entzündung 16 2.12.1 Ferritin als Akute-Phase-Protein 17 2.12.2 Serum-Eisen als Entzündungsmarker 17 3 Publikation 19 3.1 Vergleich des Eisenstatus im Serum von gesunden Fohlen und Fohlen mit Bronchopneumonie 19 4 Diskussion 30 5 Zusammenfassung 34 6 Summary 36 7 Literaturverzeichnis 38 Danksagung 47 / Introduction: Little is known about iron parameters in the blood of foals. The trace element iron is essential for mammals, its main task is the transport of oxygen. Horses take up the required iron with roughage, which has sufficiently high iron contents. The main molecule for iron storage is tissue ferritin. The serum ferritin found in the serum reflects the total amount of iron in the body. In the context of systemic inflammation, serum iron levels decrease as iron is redistributed to the tissues and migrates to the area of inflammation. There are no reference values for the parameters of iron metabolism for foals, for adult horses there is only a reference range for serum iron from 1.00-3.61 mg/L. There are no studies in which the iron parameters of foals were determined at the age of one to six months. Aim of the study: The aim of the present work was to investigate the parameters of serum iron metabolism in acute systemic inflammation in one- to six-month-old foals. In addition, it was to be determined to what extent this change in iron metabolism leads to a drop in serum iron levels. Animals, material, and methods: In this prospective study, EDTA whole blood and serum samples were collected from 66 healthy foals and 84 foals with acute systemic inflammation in the form of abscessed bronchopneumonia. The median age of the foals was 109 days. The diagnosis 'bronchopneumonia' was made by ultrasound diagnosis, the foals had a sonographically determined abscess score of more than 15cm as well as a rectally measured internal body temperature of more than 39.0 °C on the day of sampling. The healthy foals had an unremarkable clinical general examination and lung ultrasound on the day of sampling and in the previous and following week. In the laboratory, the serum amyloid A content was determined, a value > 7µg/mL confirmed the diagnosis of acute systemic inflammation. From the EDTA whole blood sample, the blood leucocyte count was determined within one hour by flow cytometry. The serum samples were analyzed in the LABOKLIN laboratory using the analyzer Cobas 8000 from Roche. Statistical analysis of the data was performed with SPSS (IBM Statistics 27). The data of the total population (n = 150) were not normally distributed. A Mann-Whitney U test was performed as a non-parametric test for two independent samples to determine significant differences between the groups. The significance level was set at p < 0.05. Results: The evaluation of the blood samples showed a median SAA level of 3.50 µg/mL for the healthy foals, the median of 541 µg/mL for the sick foals was significantly higher than in healthy foals. The determination of serum ferritin showed a median of 3.80 µg/l (IQR 2.00-4.40 µg/l) for the group of healthy foals, the median for the group of sick foals was significantly (p = 0.003) higher at 4.60 µg/l (IQR 2.48-6.40 µg/l). The serum iron values of the healthy foals were within the laboratory reference range for adult horses (median 1.57 mg/l; IQR 1.21-1.79 mg/l). The other blood iron values also corresponded to those of adult horses. The sick foals showed significantly lower serum iron levels (median 0.550 mg/l; IQR 0.368-0.778 mg/l), significantly higher ferritin and UIBC levels and significantly lower iron saturation. The evaluation of liver enzyme activities from the serum showed lower values on average for the sick foals than for the healthy foals for all measured parameters. For the trace element copper, the median of the healthy foals with 18.8 µmol/l (IQR 16.4-20.1µmol/l) is in the upper range of the reference values of 7.9-21µmol/l, the median of the sick foals with 25.7 µmol/l (IQR 23.1-29.0 µmol/l) is significantly higher than the values of the healthy foals (p < 0.001). Conclusion Acute systemic inflammation causes a drop in serum iron due to redistribution into the tissues and migration into the area of inflammation. However, since there is no evidence of an absolute iron deficiency in foals, either in the literature or in the results of this study, iron supplementation should be avoided in any case.:1 Einleitung 1 2 Literaturübersicht 2 2.1 Vorkommen und chemische Eigenschaften von Eisen 2 2.2 Eisenmetabolismus 2 2.2.1 Absorption 2 2.2.2 Transport 3 2.2.3 Speicherung, Mobilisierung 4 2.2.4 Exkretion, Verlust 5 2.3 Physiologische Funktionen 6 2.4 Feststellung des Eisenstatus 7 2.5 Eisenbedarf des Pferdes 9 2.6 Eisengehalt von Futtermitteln 10 2.7 Eisenmangel 11 2.8 Supplementation von Eisen 12 2.9 Eisenüberschuss, Toxizität 12 2.10 Bakterieller Eisenstoffwechsel 13 2.11 Entzündung 14 2.11.1 Abszedierende Bronchopneumonie 15 2.12 Eisenstoffwechsel bei systemischer Entzündung 16 2.12.1 Ferritin als Akute-Phase-Protein 17 2.12.2 Serum-Eisen als Entzündungsmarker 17 3 Publikation 19 3.1 Vergleich des Eisenstatus im Serum von gesunden Fohlen und Fohlen mit Bronchopneumonie 19 4 Diskussion 30 5 Zusammenfassung 34 6 Summary 36 7 Literaturverzeichnis 38 Danksagung 47

Master of Science / Department of Diagnostic Medicine/ Pathobiology / Lisa M. Pohlman / Background: Iron storage disease has proven to be a serious health concern for captive toucans. Physiologic mechanisms to efficiently extract iron from naturally iron-deficient diets appear the likely cause of iron overload when fed iron-sufficient diets in captivity. Iron overload can result in diabetes, heart failure, and even death. Serum ferritin concentrations are considered the most reliable screening tool to predict total body iron stores in many species, but an assay has not been available to measure serum ferritin in toucans. Objective: The purpose of this study was to develop an enzyme-linked immunosorbent assay (ELISA) to measure serum ferritin in toucans using a polyclonal antibody in a sandwich arrangement. Methods: Ferritin was isolated from toucan liver and used as a standard. A rabbit polyclonal anti-toucan antibody was used as the capture antibody and as a detection antibody conjugated to horseradish peroxidase. Linearity of toucan ferritin standards, effect of serum dilution, recovery of added ferritin standards, and intra- and inter-assay variability were determined. Results: Ferritin standards were linear from 0 to 50 ng/ml. The relationship between serum dilution and serum ferritin concentration was also linear. When 10, 20, 30, 40, or 50 ng/ml of purified toucan ferritin were added to diluted serum, the recoveries varied from 69% to 104%. The intra-assay variability for four test serum samples averaged 11% and the inter-assay variability for the same four samples averaged 11%. Conclusions: Although the results from the linearity and recovery studies are promising for assay development when viewed independently, preliminary ferritin concentrations from all toucans studied are much higher than expected. Upon further evaluation including Dot blot assays, Western blot assays, SDS-PAGE, and protein determination of the ferritin stock solution, it was determined that the ferritin stock solution did not contain a pure protein and therefore likely renders the assay invalid. Further testing is needed to confirm these findings.

No description available.

O arroz é um dos cereais mais produzidos e consumidos no mundo, cultivado em aproximadamente 156 milhões de hectares, com uma produção mundial de mais de 600 milhões de toneladas por ano. O arroz é, hoje, alimento básico para mais de dois terços da população mundial. Contudo, minerais como ferro e zinco são perdidos durante o processo de beneficiamento dos grãos para comercialização. Uma vez que a deficiência de ferro afeta cerca de três bilhões de pessoas e é a deficiência mineral mais comum em humanos, diversos esforços têm sido feitos para aumentar a concentração deste mineral em grãos de arroz. Diversos projetos têm como objetivo compreender o mecanismo de translocação de nutrientes para grãos de arroz, visando o aumento de sua concentração com fins de biofortificação do alimento. Para melhor compreender a homeostase de ferro em plantas de arroz, conduzimos experimentos para analisar possíveis funções de duas proteínas. Proteínas da família NRAMP (Natural Resistance Associated Macrophage Protein) foram descritas como tendo envolvimento na homeostase de ferro em diferentes organismos. OsNRAMP7 apresenta propriedades características da família, como os motivos DPGN e MPH, possivelmente envolvidos no transporte de metais. Oócitos de Xenopus injetados com o mRNA de OsNRAMP7 apresentaram aumento significativo na concentração de ferro. A expressão heteróloga da proteína em oócitos indica o envolvimento da proteína no transporte transmembrana de ferro. Ferritina é outra proteína envolvida na homeostase de ferro nas células. Ferritinas são proteínas esféricas, capazes de armazenar ferro no seu interior, agindo também como um estoque de ferro nas células. O armazenamento de ferro dentro desta proteína pode prevenir reações que levam a produção de radicais livres e, consequentemente, estresse oxidativo. Duas cópias do gene da ferritina foram descritas em arroz. Respostas ao estresse oxidativo em uma linhagem mutante de arroz para o gene OsFER2 foram estudadas. Quando submetidas a excesso de ferro, plantas mutantes tiveram aumento na concentração de MDA (malondialdeído) nas partes aéreas e da atividade da enzima APX (ascorbato peroxidase) em raízes, revelando respostas ao dano oxidativo quando há baixa produção de ferritina. Plantas mutantes acumulam menos biomassa do que plantas WT (wild type) mesmo em condição controle de crescimento. Isso pode indicar um possível papel da ferritina na homeostase de ferro em plantas de arroz, ainda que as mesmas não estejam em estresse por excesso de ferro. Mecanismos compensatórios como o aumento da quantidade da proteína 5 frataxina e aumento do influxo de ferro para vacúolos também devem ser investigados. Mais experimentos são necessários para melhor compreensão do papel da ferritina na homeostase de ferro em arroz. Não obstante, com os experimentos aqui apresentados é possível determinar o envolvimento da proteína OsNRAMP7 na homeostase de ferro em arroz. / Rice is one of the most produced and consumed cereals in the world, cultivated in approximately 156 million hectares, with a world production of over 600 million tons. It is a staple food for two thirds of the world population. However, minerals such as iron and zinc are lost during rice processing for commercialization. Since iron deficiency affects around three billion people, and is the most common mineral deficiency in humans, several efforts have been made in order to increase this nutrient’s levels in rice grains. Several projects have as goal to understand translocation mechanisms of nutrients to rice grains as to increase their levels for biofortification purposes. To better understand iron homeostasis in rice plants, we conducted experiments in order to analyze the putative role of two proteins. The NRAMP (Natural Resistance Associated Macrophage Protein) family was described as having an important role in iron homeostasis in different organisms. OsNRAMP7 presents characteristic features of the family, as motifs DPGN and MPH, said to be involved in metal transport. Xenopus oocytes injected with OsNRAMP7 mRNA exhibited a significant increase in iron content. Heterologous expression of the protein in oocytes indicated that the protein is involved in transmembrane iron transport. Ferritin is another protein involved in intracellular iron homeostasis. Ferritins are spherical proteins capable of storing iron in their core, also acting as an iron buffer in cells. Storage of free iron inside this protein may prevent reactions that lead to the formation of oxygen radicals and, therefore, to oxidative stress. Two ferritin genes have been described in the rice genome. We studied the oxidative stress response of a mutant line of rice with impaired expression of OsFer2. When subjected to iron excess, mutant plants increased MDA (malondialdehyde) concentration in shoots and APX (ascorbate peroxidase) enzyme activity in roots, revealing oxidative damage responses when ferritin production is impaired. Mutant plants have lower weight than WT (wild type) even in control growth condition. This may indicate a possible role of ferritin in iron homeostasis in rice plants, even when they are not under iron stress. Compensative mechanisms such as increase of frataxin levels and iron influx to the vacuole should be investigated. More experiments are required for a proper understanding of ferritin role in iron homeostasis. Still, with these experiments allowed to determine the involvement of the OsNRAMP7 protein in iron homeostasis in rice.

Iron has a dualistic character. On the one hand it is essential for the life of most organisms, on the other hand it is involved in the generation of reactive oxygen species that are implicated in diseases and aging. During evolution efficient mechanisms for uptake, handling and storage of iron in a safe way have developed to keep the balance between iron availability and minimizing the hazards. In eukaryotes, mitochondria are the central organelle for “metabolizing” iron and consequently play an important role in cellular iron homeostasis. Mitoferrins are mitochondrial carrier proteins, which are involved in iron transport into mitochondria. In vertebrates two mitoferrins exist, one (mitoferrin1) of which is essential for heme synthesis during erythropoiesis, while the function of the other (mitoferrin2) is not well defined. In the fruit fly we found only one mitoferrin gene (dmfrn), which codes most likely for a functional homologueof vertebrate mitoferrin2. In Drosophila cell culture, dmfrn overexpression resulted in an overestimation of cell sensed iron levels. The signal responsible for this, is most likely a yet unidentified compound of ISC synthesis. In the cell culture system we also showed that iron chelation blocks the progression of the cell cycle in a reversible and therefore most likely controlled way. Study of different dmfrn mutants indicates a role of dmfrn during spermatogenesis and development to adulthood. As dmfrn deletion mutants are not lethal, it is likely that other lower affinity iron transporters exist. A similar conclusion has been drawn by others from the study of yeast mitoferrin homologuemutants. Rim2p/Mrs12p has recently been implicated in mitochondrial iron transport, and might be an alternative metal carrier. We identified a putative homologuein the fruit fly and found a possible link between mutants in this gene and iron. Our results emphasize the importance of the mitochondrial iron metabolism in cellular iron homeostasis. We also show for the first time, a direct connection between the mitochondrial iron metabolism and spermatogenesis. Mutants characterized and developed by us will help to study these processes in further detail and reveal the underlying mechanisms.

No description available.

O ferro é um elemento essencial para o crescimento e desenvolvimento das plantas, envolvido em processos metabólicos essenciais, como fotossíntese e respiração. Entretanto, quando livre e em excesso, pode gerar estresse oxidativo. A toxidez por excesso de ferro trata-se do maior problema nutricional em arroz alagado, sendo responsável por perdas na produtividade. Diversas estratégias para minimizar os efeitos tóxicos do ferro vêm sendo desenvolvidas, e entre elas, o uso de cultivares tolerantes é considerada a mais efetiva. Porém, poucos dados com relação à interação entre diferentes genótipos de arroz e o ambiente encontram-se disponíveis. Utilizando-se de abordagens bioquímicas e moleculares, foram analisadas as respostas de diferentes cultivares de arroz expostas a altos níveis de ferro, crescidas em campo ou em laboratório. A toxidez por excesso de ferro teve um claro efeito foto-oxidativo, levando a quedas nos teores de clorofila, bem como a danos oxidativos. Excessivos níveis de ferro levaram a um aumento na atividade de enzimas antioxidantes, bem como a alterações no estado oxidativo da célula, modificando as concentrações das formas oxidadas e reduzidas de ascorbato e glutationa. A concentração de ferro apresentou-se variável nas cultivares tolerantes testadas. Os dados obtidos indicam que possíveis mecanismos de tolerância ao excesso de ferro podem envolver a capacidade de acumular ferro em frações superiores a 3kDa, maior atividade de SOD (através da expressão diferencial de três isoformas), bem como a limitação da captura do metal, possivelmente envolvendo a lignificação e remodelamento da parede celular das células da raiz. Altos níveis de ferro levaram ao acúmulo de transcritos dos genes de ferritina, em especial de OsFER2, dependente de um passo oxidativo, bem como à expressão de outros genes relacionados a homeostase de ferro. Cinco genes pertencentes às famílias gênicas ZIP (OsZIP1, OsZIP7 e OsZIP8) e NRAMP (OsNRAMP4 e OsNRAMP5) tiveram sua expressão induzida em plantas expostas a altos níveis de ferro, sugerindo seu possível envolvimento em respostas ao excesso de ferro. Os genes OsIRT1 e OsIRT2, OsNRAMP1 e OsYS7, cuja expressão relativa foi aumentada em condições de deficiência de ferro, tiveram sua expressão reduzida em excesso de ferro. Esses genes codificam transportadores de alta afinidade por ferro, sugerindo a ocorrência de uma resposta coordenada, dependente da concentração de ferro. Plantas de cultivares de arroz distintas apresentaram diferentes mecanismos de tolerância ao excesso de ferro. / Iron is an essential nutrient for growth and development of plants, involved in important plant biological processes, such as photosynthesis and respiration. However, when free and in excessive levels inside the cell, iron can act as a pro-oxidant, leading to oxidative stress. Iron toxicity is considered the major nutritional disorder in waterlogged and lowland rice, being responsible for losses on rice production. Several management strategies have been developed to overcome iron toxicity, and the most cost-effective approach is the use of tolerant rice cultivars. Despite this, few data concerning the relation between different rice cultivars and its environment are available. Through the use of molecular and biochemical approaches, we analyzed the responses of distinct rice genotypes exposed to iron excess, cultivated in the field or in the laboratory. Iron toxicity had a clear photo oxidative damage, leading to decreases in chlorophyll levels and generating oxidative damage. Iron excess also induced the activity of antioxidant enzymes, as well as an alteration in the redox status of the cell, besides concentration varied between the studied cultivars. Mechanisms involved in the tolerance to iron toxicity may involve the capacity to accumulate iron at molecular mass fractions, a higher SOD activity (probably through the differential induction of SOD isoforms), and also the limitation of iron uptake in nutrient solution. This limitation may rely in the root cell wall remodeling and lignifications. Iron lead to an up-regulation of ferritin genes, especially OsFER2, with this induction being dependent on an oxidative step. Iron excess also lead to an induction on the relative gene expression of iron homeostasis-related genes. Five genes belonging to two distinct gene families, ZIP (OsZIP1, OsZIP7 and OsZIP8) and NRAMP (OsNRAMP4 e OsNRAMP5), were up-regulated in plants exposed to iron excess, suggesting their possible role in response to excessive amounts of iron. Interestingly, five genes (OsIRT1 and OsIRT2, OsNRAMP1 and OsYS7) up-regulated by iron deficiency, were regulated in an opposite way by iron excess. All the five genes encode proteins involved in the uptake and transport of iron, suggesting a coordinated response, depending on the iron concentration. Taken together, our results indicated that different rice cultivars can use distinct tolerance mechanisms.

O arroz é um dos cereais mais produzidos e consumidos no mundo, cultivado em aproximadamente 156 milhões de hectares, com uma produção mundial de mais de 600 milhões de toneladas por ano. O arroz é, hoje, alimento básico para mais de dois terços da população mundial. Contudo, minerais como ferro e zinco são perdidos durante o processo de beneficiamento dos grãos para comercialização. Uma vez que a deficiência de ferro afeta cerca de três bilhões de pessoas e é a deficiência mineral mais comum em humanos, diversos esforços têm sido feitos para aumentar a concentração deste mineral em grãos de arroz. Diversos projetos têm como objetivo compreender o mecanismo de translocação de nutrientes para grãos de arroz, visando o aumento de sua concentração com fins de biofortificação do alimento. Para melhor compreender a homeostase de ferro em plantas de arroz, conduzimos experimentos para analisar possíveis funções de duas proteínas. Proteínas da família NRAMP (Natural Resistance Associated Macrophage Protein) foram descritas como tendo envolvimento na homeostase de ferro em diferentes organismos. OsNRAMP7 apresenta propriedades características da família, como os motivos DPGN e MPH, possivelmente envolvidos no transporte de metais. Oócitos de Xenopus injetados com o mRNA de OsNRAMP7 apresentaram aumento significativo na concentração de ferro. A expressão heteróloga da proteína em oócitos indica o envolvimento da proteína no transporte transmembrana de ferro. Ferritina é outra proteína envolvida na homeostase de ferro nas células. Ferritinas são proteínas esféricas, capazes de armazenar ferro no seu interior, agindo também como um estoque de ferro nas células. O armazenamento de ferro dentro desta proteína pode prevenir reações que levam a produção de radicais livres e, consequentemente, estresse oxidativo. Duas cópias do gene da ferritina foram descritas em arroz. Respostas ao estresse oxidativo em uma linhagem mutante de arroz para o gene OsFER2 foram estudadas. Quando submetidas a excesso de ferro, plantas mutantes tiveram aumento na concentração de MDA (malondialdeído) nas partes aéreas e da atividade da enzima APX (ascorbato peroxidase) em raízes, revelando respostas ao dano oxidativo quando há baixa produção de ferritina. Plantas mutantes acumulam menos biomassa do que plantas WT (wild type) mesmo em condição controle de crescimento. Isso pode indicar um possível papel da ferritina na homeostase de ferro em plantas de arroz, ainda que as mesmas não estejam em estresse por excesso de ferro. Mecanismos compensatórios como o aumento da quantidade da proteína 5 frataxina e aumento do influxo de ferro para vacúolos também devem ser investigados. Mais experimentos são necessários para melhor compreensão do papel da ferritina na homeostase de ferro em arroz. Não obstante, com os experimentos aqui apresentados é possível determinar o envolvimento da proteína OsNRAMP7 na homeostase de ferro em arroz. / Rice is one of the most produced and consumed cereals in the world, cultivated in approximately 156 million hectares, with a world production of over 600 million tons. It is a staple food for two thirds of the world population. However, minerals such as iron and zinc are lost during rice processing for commercialization. Since iron deficiency affects around three billion people, and is the most common mineral deficiency in humans, several efforts have been made in order to increase this nutrient’s levels in rice grains. Several projects have as goal to understand translocation mechanisms of nutrients to rice grains as to increase their levels for biofortification purposes. To better understand iron homeostasis in rice plants, we conducted experiments in order to analyze the putative role of two proteins. The NRAMP (Natural Resistance Associated Macrophage Protein) family was described as having an important role in iron homeostasis in different organisms. OsNRAMP7 presents characteristic features of the family, as motifs DPGN and MPH, said to be involved in metal transport. Xenopus oocytes injected with OsNRAMP7 mRNA exhibited a significant increase in iron content. Heterologous expression of the protein in oocytes indicated that the protein is involved in transmembrane iron transport. Ferritin is another protein involved in intracellular iron homeostasis. Ferritins are spherical proteins capable of storing iron in their core, also acting as an iron buffer in cells. Storage of free iron inside this protein may prevent reactions that lead to the formation of oxygen radicals and, therefore, to oxidative stress. Two ferritin genes have been described in the rice genome. We studied the oxidative stress response of a mutant line of rice with impaired expression of OsFer2. When subjected to iron excess, mutant plants increased MDA (malondialdehyde) concentration in shoots and APX (ascorbate peroxidase) enzyme activity in roots, revealing oxidative damage responses when ferritin production is impaired. Mutant plants have lower weight than WT (wild type) even in control growth condition. This may indicate a possible role of ferritin in iron homeostasis in rice plants, even when they are not under iron stress. Compensative mechanisms such as increase of frataxin levels and iron influx to the vacuole should be investigated. More experiments are required for a proper understanding of ferritin role in iron homeostasis. Still, with these experiments allowed to determine the involvement of the OsNRAMP7 protein in iron homeostasis in rice.

Made available in DSpace on 2014-06-11T19:32:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T20:43:03Z : No. of bitstreams: 1 estevao_if_dr_sjrp_parcial.pdf: 122795 bytes, checksum: 747e86122e594e13887a3ebfff701723 (MD5) Bitstreams deleted on 2015-08-07T12:19:53Z: estevao_if_dr_sjrp_parcial.pdf,. Added 1 bitstream(s) on 2015-08-07T12:20:55Z : No. of bitstreams: 1 000642193.pdf: 2022251 bytes, checksum: 43e74970fe45b54e28f7ac29e1ec9d71 (MD5) / A beta talassemia heterozigota pode ser decorrente de mutações β 0 ou β + , de acordo com ausência ou redução na produção das cadeias da beta globina, respectivamente. Poucos estudos na literatura correlacionam possíveis efeitos deletérios dessas mutações, quando em heterozigose. A coerança com mutações em genes, não localizados no cluster do gene da β-globina, podem modificar o fenótipo talassêmico. O estresse oxidativo gerado pelo aumento de ERO é um dos mecanismos responsáveis pala aceleração da apoptose celular. Há evidências que o excesso de cadeias alfa, mesmo em pequenas quantidades, seja suficiente para iniciar a geração de ERO e, portanto, estresse oxidativo em beta talassêmicos heterozigotos. Nas formas homozigotas ou com dupla heterozigose os pacientes apresentam anemia grave, transfusão dependente, e a sobrecarga de ferro é uma das mais frequentes e graves complicações nesses indivíduos. Na forma heterozigota a ocorrência de sobrecarga de ferro é rara, porém alterações no perfil do ferro são observadas. O objetivo desse trabalho foi analisar a influência dos níveis de ferritina sérica, das mutações no gene da β-globina, das mutações no gene HFE, da atividade das enzimas de detoxificação GST e CAT e dos polimorfismos das GSTs na expressão fenotípica de indivíduos beta talassêmicos heterozigotos. Foram avaliados 158 portadores de beta talassemia heterozigota diagnosticados previamente por metodologias clássicas. Todos adultos, de origem caucasóide, residentes na região Central do Estado de São Paulo e acompanhados em serviços públicos ou privados da região. O grupo controle foi composto de 24 adultos, caucasóides e residentes na mesma região. A avaliação hematológica foi realizada pelo hemograma, em contadores automatizados de células e quantificação das sub frações de Hb A2 e Hb F por HPLC. O perfil... / Beta thalassemia may occur due to mutations β 0 or β +, according to absence or reduction in the production of beta globin chains, respectively. Few studies have been done correlating possible deleterious effects of these mutations, when in heterozygous. Co-inheritance with mutations in the genes, not located in the cluster of β-globin gene may modify the phenotype of the thalassemic. Stress oxidative generated by the increase of ROS is one of the mechanisms responsible for the acceleration of cell apoptosis. There are evidences that alpha chains in excess, even in small quantities, are sufficient to start ROS generation and thus oxidative stress in heterozygous beta thalassemic. In homozygous form or genetic heterozygous compound present severe anemia, transfusion dependent and iron overload is one of the complications more frequent and severe in these patients. In the heterozygous form the presence of iron overload is rare, but alterations in the profile of iron are observed. The aim of this study was to examine influence of the levels of serum ferritin, the mutations in the β-globin gene, the mutations in the HFE gene, activity of enzymes detoxification, GST and CAT, and polymorphisms of GSTs in the expression phenotypic of individuals heterozygous beta thalassemic. We evaluated 158 patients with beta thalassemia heterozygous previously diagnosed by classic methodologies. They were all adults, Caucasian origin, residents in the Central Region of São Paulo state and accompanied in public or private services of the region. The control group consisted of 24 adults, Caucasians and residents in the same region. Hematologic evaluation was carried out by hemogram, in automated counters of cells and quantification of the subfractions of Hb A2 and Hb F by HPLC; the iron profile was based on the transferrin saturation , calculated on the serum iron and total iron binding capacity... (Complete abstract click electronic access below)