Le fer est un élément présent en abondance dans la croûte terrestre, indispensable à la quasi-totalité des êtres vivants. Cependant, en milieu marin la biodisponibilité du fer est souvent faible et sporadique. Les micro-algues du phytoplancton ont développé des stratégies pour faire face à cette limitation en fer et s’adapter à des niches écologiques variables. Les micro-algues vertes du genre Ostreococcus (Prasinophyceae) présentent une large distribution géographique dans l’océan mondial, et de nombreux écotypes venant de milieux contrastés ont été isolés. L’objectif principal de ma thèse était d’étudier les différentes stratégies mises en place par le genre Ostreococcus, et notamment l’influence de la lumière et de l’horloge circadienne, dans la gestion de la carence en fer. Mon travail s’est focalisé sur l’étude d’Ostreococcus tauri, écotype lagunaire (Clade C), que de récentes techniques de transformation par insertion et recombinaison homologue ont promu comme un organisme modèle pour des approches de génétique fonctionnelle. J’ai étudié la ferritine, une protéine impliquée dans la gestion de la réserve en fer chez de nombreux organismes, et mis en évidence sa régulation par l’alternance jour/nuit et l’horloge circadienne. J’ai montré son rôle dans l’assimilation du fer, la régulation de l’homéostasie du fer et le recyclage du fer intracellulaire lors d’une carence. Enfin, j’ai caractérisé les stratégies d’acclimatation et d’adaptation à la carence en fer chez plusieurs écotypes d’Ostreococcus, dont O. tauri, RCC 802 (Clade A), RCC 809 (Clade B) et un mutant de taille/biomasse. Une stratégie d’acclimatation par réduction de la biomasse cellulaire a été mise en évidence. / Iron is an abundant element in the earth crust and is essential for almost organisms. In the marine environment, however, its bioavailability is often low and the iron supplies sporadic. Phytoplanktonic species have developed various strategies to face iron limitation and adapt to different ecological niches. Green picoalgae from the genus Ostreococcus (Prasinophyceae) are widespread in the global ocean and numerous ecotypes have been isolated from contrasted environments. The main objective of my thesis was to identify the strategie(s) used by the genus Ostreococcus in response to iron starvation and in particular the influence of the day/night cycle and the circadian clock in the regulation of iron homeostasis. I focused my work on the lagoon ecotype, Ostreococcus tauri (Clade C), which has emerged as a model organism for functional genomics approaches thanks to the development of genetic transformation by random insertion and homologous recombination. I have studied ferritin, a protein involved in iron storage which is present throughout the tree of life. I showed that ferritin is regulated by the light/dark cycle and the circadian clock and that it is a key player in the regulation of iron uptake and the recycling. Finally, I characterized the acclimation and adaptation strategies to iron limitations of several Ostreococcus ecotypes including O.tauri, RCC802 (Clade A), RCC809 (Clade B) and a cell biomass mutant of O.tauri. The reduction of cell biomass appears to be a main mechanism of acclimation in response to iron limitation.

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.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A infecÃÃo pelo Helicobacter pylori à uma das infecÃÃes mais prevalentes do mundo. Existem fortes evidÃncias de que a transmissÃo dessa bactÃria ocorre na infÃncia, e estudos epidemiolÃgicos em crianÃas sÃo essenciais para que se entenda os aspectos relacionados com a dinÃmica da infecÃÃo e suas conseqÃÃncias. Previamente pesquisadores vinculados ao LaboratÃrio de Gastroenterologia da Universidade Federal do Cearà avaliaram a prevalÃncia de H.pylori em crianÃas atà quatorze anos de idade em uma comunidade fixa de baixa renda, denominada Parque UniversitÃrio. No presente estudo, foi realizado outro corte transversal aproximadamente oito anos apÃs a anÃlise inicial nesse mesmo grupo de crianÃas, com o objetivo de avaliar a dinÃmica da infecÃÃo e se as crianÃas infectadas sÃo mais susceptÃveis a terem anemia, deficiÃncia de ferrritina, folato e vitamina B12 do que as nÃo infectadas. A infecÃÃo por H. pylori foi avaliada em 133 crianÃas por meio do teste respiratÃrio, previamente validado para a populaÃÃo brasileira. Foram coletadas amostras de sangue em 108 crianÃas para a realizaÃÃo de hemograma e a dosagem de concentraÃÃes sÃricas de ferritina, vitamina B12 e folato por meio da tÃcnica de quimioluminescencia utilizando Immulite  2000. A prevalÃncia da infecÃÃo por H. pylori nas crianÃas foi de 53,4% (71/133) em 2000, enquanto a prevalÃncia de H. pylori nas mesmas crianÃas em 2008 foi de 64,7% (86/133). ApÃs 8 anos, a maioria (88,7%) das crianÃas H. pylori positivas permaneceram infectadas. A taxa de aquisiÃÃo da infecÃÃo por H. pylori apÃs 8 anos da anÃlise inicial foi de 17,3% (23/133) e a taxa de cura espontÃnea da infecÃÃo foi de 6% (8/133). As taxas de aquisiÃÃo foram maiores em crianÃas com idade de 10 anos ou menos, atingindo 30,2% em crianÃas menores de 5 anos. A prevalÃncia da infecÃÃo em crianÃas com menos de 5 anos aumentou de 39.6% em 2000 para 62,2% em 2008 (p=0,020). NÃo houve diferenÃa estatisticamente significante entre anemia, concentraÃÃes sÃricas de ferritina, vitamina B12 e folato em indivÃduos H. pylori positivos comparados aos H. pylori negativos. Em conclusÃo, a infecÃÃo por H. pylori à adquirida precocemente na infÃncia, com altas taxas de aquisiÃÃo em crianÃas com 10 anos ou menos. NÃo foi encontrado associaÃÃo entre a infecÃÃo por H. pylori e anemia, nÃveis sÃricos de vitamina B12, ferritina e folato / A infecÃÃo pelo Helicobacter pylori à uma das infecÃÃes mais prevalentes do mundo. Existem fortes evidÃncias de que a transmissÃo dessa bactÃria ocorre na infÃncia, e estudos epidemiolÃgicos em crianÃas sÃo essenciais para que se entenda os aspectos relacionados com a dinÃmica da infecÃÃo e suas conseqÃÃncias. Previamente pesquisadores vinculados ao LaboratÃrio de Gastroenterologia da Universidade Federal do Cearà avaliaram a prevalÃncia de H.pylori em crianÃas atà quatorze anos de idade em uma comunidade fixa de baixa renda, denominada Parque UniversitÃrio. No presente estudo, foi realizado outro corte transversal aproximadamente oito anos apÃs a anÃlise inicial nesse mesmo grupo de crianÃas, com o objetivo de avaliar a dinÃmica da infecÃÃo e se as crianÃas infectadas sÃo mais susceptÃveis a terem anemia, deficiÃncia de ferrritina, folato e vitamina B12 do que as nÃo infectadas. A infecÃÃo por H. pylori foi avaliada em 133 crianÃas por meio do teste respiratÃrio, previamente validado para a populaÃÃo brasileira. Foram coletadas amostras de sangue em 108 crianÃas para a realizaÃÃo de hemograma e a dosagem de concentraÃÃes sÃricas de ferritina, vitamina B12 e folato por meio da tÃcnica de quimioluminescencia utilizando Immulite  2000. A prevalÃncia da infecÃÃo por H. pylori nas crianÃas foi de 53,4% (71/133) em 2000, enquanto a prevalÃncia de H. pylori nas mesmas crianÃas em 2008 foi de 64,7% (86/133). ApÃs 8 anos, a maioria (88,7%) das crianÃas H. pylori positivas permaneceram infectadas. A taxa de aquisiÃÃo da infecÃÃo por H. pylori apÃs 8 anos da anÃlise inicial foi de 17,3% (23/133) e a taxa de cura espontÃnea da infecÃÃo foi de 6% (8/133). As taxas de aquisiÃÃo foram maiores em crianÃas com idade de 10 anos ou menos, atingindo 30,2% em crianÃas menores de 5 anos. A prevalÃncia da infecÃÃo em crianÃas com menos de 5 anos aumentou de 39.6% em 2000 para 62,2% em 2008 (p=0,020). NÃo houve diferenÃa estatisticamente significante entre anemia, concentraÃÃes sÃricas de ferritina, vitamina B12 e folato em indivÃduos H. pylori positivos comparados aos H. pylori negativos. Em conclusÃo, a infecÃÃo por H. pylori à adquirida precocemente na infÃncia, com altas taxas de aquisiÃÃo em crianÃas com 10 anos ou menos. NÃo foi encontrado associaÃÃo entre a infecÃÃo por H. pylori e anemia, nÃveis sÃricos de vitamina B12, ferritina e folato / Helicobacter pylori infection is one the most prevalent infections in the world. There is a strong evidence that it is acquired in childhood. Therefore epidemiological studies in children are essential to understand the dynamics of this infection and also to evaluate the possible consequences of H. pylori infection in children. Previously it was evaluated the prevalence of H. pylori in children up to fourteen years of age at a low-income community called Parque UniversitÃrio. The present study is a cohort of a group of children eight years after the first study, with the aim of evaluating the dynamics of H. pylori infection and also to check if infected children are more likely to have anemia and ferrritin, folate and vitamin B12 deficiency when compared to non-infected children. The infection was evaluated in 133 individuals through the 13C urea breath test, previously validated for the brazilian population. Blood was sampled from 108 individuals to dose levels of ferritin, vitamin B12 and folate by chemiluminescence using Immulite  2000 and hemoglobin. The prevalence of H. pylori infection in the individuals was 53.4% (71/133) in 2000, while the prevalence of H. pylori in the same group in 2008 was 64.7% (86/133). It was found that after eight years, the majority (88.7%) of H. pylori positive individuals remained positive for infection. The rate of acquisition of infection by H. pylori after 8 years of the initial analysis was 17.3% (23/133) and the rate of loss of infection was 6% (8/133). The acquisition rates were higher in children younger than 10 years, reaching 30.2% in children younger than 5 years. The prevalence of infection in children under 5 years increased from 39.6% in 2000 to 62.2% in 2008 (p = 0.020). There was no statistically significant difference between anemia, serum ferritin, vitamin B12 and folate in H. pylori-positive individuals compared to H. pylori-negative. In conclusion, H. pylori infection is acquired early in childhood, with high rates of acquisition in children under 10 years. There was no association between H. pylori infection and anemia, serum vitamin B12 and folate levels among the individuals studied. / Helicobacter pylori infection is one the most prevalent infections in the world. There is a strong evidence that it is acquired in childhood. Therefore epidemiological studies in children are essential to understand the dynamics of this infection and also to evaluate the possible consequences of H. pylori infection in children. Previously it was evaluated the prevalence of H. pylori in children up to fourteen years of age at a low-income community called Parque UniversitÃrio. The present study is a cohort of a group of children eight years after the first study, with the aim of evaluating the dynamics of H. pylori infection and also to check if infected children are more likely to have anemia and ferrritin, folate and vitamin B12 deficiency when compared to non-infected children. The infection was evaluated in 133 individuals through the 13C urea breath test, previously validated for the brazilian population. Blood was sampled from 108 individuals to dose levels of ferritin, vitamin B12 and folate by chemiluminescence using Immulite  2000 and hemoglobin. The prevalence of H. pylori infection in the individuals was 53.4% (71/133) in 2000, while the prevalence of H. pylori in the same group in 2008 was 64.7% (86/133). It was found that after eight years, the majority (88.7%) of H. pylori positive individuals remained positive for infection. The rate of acquisition of infection by H. pylori after 8 years of the initial analysis was 17.3% (23/133) and the rate of loss of infection was 6% (8/133). The acquisition rates were higher in children younger than 10 years, reaching 30.2% in children younger than 5 years. The prevalence of infection in children under 5 years increased from 39.6% in 2000 to 62.2% in 2008 (p = 0.020). There was no statistically significant difference between anemia, serum ferritin, vitamin B12 and folate in H. pylori-positive individuals compared to H. pylori-negative. In conclusion, H. pylori infection is acquired early in childhood, with high rates of acquisition in children under 10 years. There was no association between H. pylori infection and anemia, serum vitamin B12 and folate levels among the individuals studied.

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.

Diploma thesis deals with the study of dissociation and reassociation of ferritin protein cages and their use in nanomedicine. Most studies that are focused on targeted transport of pharmaceuticals using ferritin cages work with horse spleen ferritin. It is, however, its origin, which leads to increasingly frequent questions about possible immunogenicity in the patient's organism, which also provides the main motivation to test the possibility of encapsulation of low-molecular drugs into ferritins originating from alternative organisms. In the practical part the method for the study of dissociation was experimentally designed. Native polyacrylamide gel electrophoresis was used to study dissociation of equine ferritin composed of different subunit, human ferritin, and archeal Pyrococcus furiosus ferritin. The obtained subunit dissociation results were used to encapsulate the low molecular chemotherapeutic drug doxorubicin and for further characterization of the ferritin-doxorubicin complex. The efficacy of the designed nanoformulations has been verified in the treatment of malignant breast cancer. Human ferritin proves to be the optimal one. Its composition of heavy subunits corresponds to a lower protein stability, thus a more efficient opening of the structure and consequent encapsulation of the cytostatics occurs. With its 60% encapsulation efficiency of doxorubicin, low polydispersity index, effective cytotoxicity of ferritin-doxorubicin complex and minimal risk of immune response to the patient's organism, human ferritin achieves better results than commonly used horse spleen ferritin.

Active learning strategies are important for facilitating deep learning that may be carried throughout life, but which is still finding its way into the college setting. Educators are not often trained in effective learning practices, which reduces the cognitive and proficiency gains of their students. By providing such guidance in the formative years of a teacher’s training, we hypothesize that the learning environment will be greatly enriched and enhanced. On the opposite end of the spectrum of life and cognition, the plague of dementia also warrants examination. Alzheimer’s disease (AD), an incurable neurodegenerative disorder progressing from the medial temporal lobe, is the most common form of dementia diagnosed in people over age 65, afflicting 30-40% of those 85 years and older. Despite its prevalence, effective treatments are limited because the principal causes and triggers of AD are not entirely understood. Growing evidence demonstrates that oxidative stress (OS) is an important factor contributing to the initiation and progression of AD. A key player contributing to this OS is iron, an essential trace mineral which is required for proper neuronal function, but which generates reactive oxygen species during redox transitions. Intracellular labile iron pool (LIP) levels are strictly regulated by proteins such as transferrin (import), ferroportin (export), and ferritin (storage). However, when these proteins become dysregulated, excess iron associates with other proteins such as amyloid beta (Aβ) and tau, aggregations of which are hallmarks of AD. In our hypothetical model, under extensive or prolonged OS, as occurs in AD, much larger Aβ plaques form because the stress does not abate. Hyperphosphorylated tau is the last resort to protect the cell against free iron, and aggregates when the LIP is elevated because neither iron storage in ferritin nor iron export through ferroportin can relieve the neurons of the free iron.

Iron deficiency is common in frequent blood donors. At Ryhov blood center, hemoglobin and ferritin levels are measured in all newly registered blood donors. After June 2017, additional control for S-ferritin levels was added after the 4th and 5th blood donation, in women and men, respectively. The study aimed to evaluate S-ferritin levels in newly registered blood donors in 2018 and compare the number of rejected donors between men and women. Moreover, to compare S-ferritin levels between new registration and follow-up and compare the number of screening controls in 2016 with 2020. The study included all newly registered and rejected blood donors in 2018. Additionally, blood donors who were newly registered during 2018–2019 and underwent the follow-up S-ferritin control between 2018–2021 were included. The number of newly registered in 2018 was 544 blood donors, 17% were rejected due to low S-ferritin levels where all were women. Results showed that S-ferritin levels decreased at follow-up and the difference was greater in men than women. However, women had lower S-ferritin levels at new registration compared to men. The study could not show that follow-up S-ferritin control has an effect in reducing the number of screenings controls.

Ferritin is the major protein responsible for the sequestration, storage and release of intracellular iron. The ferritin protein shell exists as heteropolymers of various combinations of two types of subunits, the H-subunit and L-subunit, a phenomenon that gives rise to the existence of isoferritins. As the roles of the H-subunit and L-subunit differ in the mineralization process, the subunit composition of ferritin will influence the metabolic properties of the assembled ferritin molecules. The primary aim of the present study was to quantitatively measure the expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron in patients with chronic T-helper cell type-1 immune stimulation. A second aim was to investigate the possible role that the expression of the H-subunit and L-subunit of ferritin may have in the establishment and maintenance of an iron transfer block. The study subjects included 48 patients with chronic diseases from the Department of Internal Medicine, Kalafong Hospital and 10 patients with osteoarthritis, scheduled for hip replacement at the Department of Orthopaedics, Pretoria Academic Hospital. Bone marrow and blood samples were collected from each patient. The expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron was quantitatively evaluated by post-embedding immunolocalisation with immunogold transmission electron microscopy. The patients were subdivided into groups with a predominantly T-helper cell type-1 immune reaction (pro-inflammatory) and normal immune status on the basis of C-reactive protein, neopterin and cytokines (INF-γ, TNF-α, Il-1β, Il-6, Il-12, Il-2, Il-8, GM-CSF, Il-4, Il-5, TGF-β and Il-10). The study showed • up-regulation of the H-subunit of ferritin in the bone marrow macrophage in patients with chronic T-helper cell type-1 immune stimulation • no effects for chronic T-helper cell type-1 immune stimulation on the expression of the L-subunit of ferritin in the bone marrow macrophage • no effects for chronic T-helper cell type-1 immune stimulation on the expression of either the H-subunit or L-subunit of ferritin in cells of the bone marrow erythron • a 70% prevalence of iron transfer block in patients with chronic T-helper cell type-1 immune stimulation • up-regulation of the H-subunit of ferritin in the bone marrow macrophage in osteoarthritis patients who had normal T-helper cell type-1 immune activity, but significantly increased TGF-β levels • up-regulation of the H-subunit of ferritin in the patients with iron transfer block • iron availability loses its primary role in the establishment of the circulating red blood profile in conditions with chronic pro-inflammatory activity • indications that the H-subunit and L-subunit of ferritin may play a role in the iron availability for red blood cell haemoglobin production • various correlations in the osteoarthritis patients between the H-subunit and L-subunit of ferritin and different cytokines / Thesis (PhD)--University of Pretoria, 2009. / Physiology / unrestricted

Ferritin is an iron storage protein consisting of H and L chains to form a 24-subunit heropolymer. Ceruloplamin oxidizes Fe(II) and then loads the iron into ferritin. This research was conducted to determine which ferritin subunit is involved and whether a proposed iron-loading channel is required for iron loading by ceruloplasmin. Recombinant rat liver H and L chain ferritin homopolymers, designated as rH-Ft and rL-Ft, respectively, were produced using insect cell-baculovirus and Escherichia coli expression systems. The expressed rH-Ft strongly suppressed the growth of the host. The rH-Ft expressed in the E. coli contained approximately 150 iron atoms/ferritin and was observed to have protein damage, which was found to affect iron-loading by ceruloplasmin. The ferritin expressed in the E. coli system apparently was not proper for this iron loading study. Alternatively, the ferritins expressed in the insect cell-baculovirus system were utilized for this purpose . Ceruloplasmin was able to load iron into the rH-Ft, but not the rL-Ft. The initial rate of loading iron into the rH-Ft by ceruloplasmin was similar to that of native rat liver ferritin heteropolymer. Both the rH-Ft and the native rat liver ferritin could be maximally loaded with iron by ceruloplasmin up to 2,500 iron atoms/ferritin. When the rH-Ft or the native ferritin was present, the ferroxidase activity of ceruloplasmin was enhanced. No such enhancement was observed in the presence of the rL-Ft. This suggests that ceruloplasmin only associates with the ferritin H, but not L, chain during iron loading. The role of an a-helix bundle channel in iron loading by ceruloplasmin was investigated by using sitedirected mutagenesis. The channel in the rH-Ft was closed by mutation E62K and H65G to form a K62 to El07 salt bridge, which is thought to exist in the L chain. Conversely, the salt bridge in the channel of the L chain was removed by mutation K58E and G61H to form a channel similar to that in the four-a-helix bundle of the H chain. The initial rate of loading iron into the rL-FT mutant by ceruloplasmin was 50% of that for loading iron into the rH-Ft. When 500 atoms of iron per ferritin were used for loading, 98% loaded into the rH-Ft by ceruloplasmin in 5 minutes, but only 30% loaded into the rL-Ft mutant in the same time. The ferroxidase activity of ceruloplasmin was enhanced in the presence of the rHFt and its mutant, but not in the presence of the rL-Ft or its mutant. These results indicate that the association of ceruloplasmin and ferritin is required and the a-helix bundle channel is a channel for iron loading.