Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [100]-125).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [115]-144).

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 105-114.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 121-135).

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

My thesis has accomplished 3 significant contributions to neuroscience. Firstly, I have discovered a novel example of vertebrate deep-brain photoreception. Spontaneously generated fictive locomotion from the isolated nervous system of pro-metamorphic Xenopus tadpoles is sensitive to the ambient light conditions, despite input from the classical photoreceptive tissues of the retina and pineal complex being absent. The photosensitivity is found to be tuned to short wavelength UV light and is localised to a small region of the caudal diencephalon. Within this region, I have discovered a population of neurons immuno-positive for a UV-specific opsin protein, suggesting they are the means of phototransduction. This may be a hitherto overlooked mechanism linking environmental luminance to motor behaviour. Secondly, I have advanced the collective knowledge of how both nitric oxide and dopamine contribute to neuromodulation within motor control systems. Nitric oxide is shown to have an excitatory effect on the occurrence of spontaneous locomotor activity, representing a switch in its role from earlier in Xenopus development. Moreover, this excitatory effect is found to be mediated in the brainstem despite nitric oxide being shown to depolarise spinal neurons. Thirdly, I have developed a new preparation for patch-clamp recording in pro-metamorphic Xenopus tadpoles. My data suggest there are several changes to the cellular properties of neurons in the older animals compared with the embryonic tadpole; there appears to be an addition of Ih and K[sub](Ca) channels and the presence of tonically active and intrinsically rhythmogenic neurons. In addition, I have shown that at low doses dopamine acts via D2-like to hyperpolarise the membrane potential of spinal neurons, while at higher doses dopamine depolarises spinal neurons. These initial data corroborate previously reported evidence that dopamine has opposing effects on motor output via differential activation of dopamine receptor subtypes in Xenopus tadpoles.

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.

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.

Mon projet de thèse s’inscrit dans le cadre des études visant à comprendre comment les cellules embryonnaires intègrent les différents signaux auxquels elles sont exposées pour s’engager dans une voie de différenciation définie. Il est plus particulièrement centré sur le rôle des protéines Smad dans ces processus et peut se diviser en deux axes de recherche. Le premier a trait au rôle de Mad (Smad1) dans les interactions entre signaux Wnt (Wg) et BMP chez la drosophile. Nous avons pu démontrer que la forme Mad non phosphorylée par le récepteur BMP se lie au complexe transcriptionnel ß-catenin/dTCF et est requise pour le signal Wnt canonique. La phosphorylation de Mad par le récepteur BMP dirige Mad vers la voie BMP, créant la possibilité d’une compétition entre ces deux classes de signaux. Le second axe de recherche concerne le facteur de transcription Smad4 qui est requis pour la transduction des signaux TGF-ß et BMP. J’ai pu identifier trois sites potentiels de phosphorylation par la kinase GSK3 dans la séquence primaire de Smad4. En utilisant de nombreuses techniques de biochimie, j’ai pu montrer que Smad4 est phosphorylé par la kinase Erk, puis par GSK-3 en réponse à un signal FGF. Lorsque Smad4 est doublement phosphorylé, il est reconnu par une E3-ligase, beta-TrCP, ce qui entraine sa polyubiquitination et sa dégradation. La voie Wnt étant capable d’inhiber GSK-3, j’ai pu montrer que Smad4 est stabilisé par des signaux Wnt. Ce mécanisme augmente la sensibilité des cellules aux signaux TGF-beta lorsqu’elles reçoivent également un signal Wnt. / During my PhD I studied how cells receive and integrate multiple signals from the extracellular milieu. I focused on Smad proteins and my project can be divided into two parts. My first project was centered on the transcription factor Mad (Smad1) and its requirement for the BMP and Wg pathways. Using a combination of genetic and biochemistry experiments, we showed that Mad is required for Wg signaling both in Tcf reporter gene assays and in vivo in Drosophila. We found that the choice for Mad to transduce Dpp or Wg signals is controlled by C-terminal phosphorylations so that Mad binds to Pangolin and participates in Wg target genes transcription only when not phosphorylated at its C-terminus. This results in a competition between Dpp and Wg controlled by the phosphorylation state of Mad. My second project was focused on the tumor suppressor Smad4. When I first joined the lab, I identified three new potential GSK3 phosphorylation sites in Smad4 primary sequence. I used a home-made phospho-specific antibody to demonstrate that FGF or EGF stimulation trigger Erk-mediated phosphorylation of Smad4 which primes subsequent GSK3 phosphorylations. These phosphorylations regulate a transcription activation domain located in Smad4 linker region and generate a Wnt-regulated phosphodegron recognized by the E3 ligase beta-TrCP. This mechanism provides a means of integrating distinct pathways which would otherwise remain insulated, allowing cells to sense FGF and Wnt inputs and adapt TGF-beta outcome to their context. It provides a molecular explanation of the long-standing mystery of the “competence modifier” effect of Wnt on Nodal signals discovered 20 years ago.

No description available.