Memoria para optar al Título Profesional de Medico Veterinario / Los estudios de privación de REM típicamente apuntan a la homeostasis de largo plazo, por lo cual se han llevado a cabo privaciones de larga duración de REM, las cuales tenían una prolongación en el tiempo de algunas horas hasta incluso días. No obstante existe también la homeostasis de corto plazo para la cual no se han hecho estudios de privación. Es por esto que se utilizaron 12 ratas Sprague Dawley machos, las cuales fueron estudiadas polisomnograficamente, luego de una habituación a condiciones de laboratorio. Fueron mantenidas bajo condiciones de luz: oscuridad constante 12:12, con agua y alimentación Ad-libitum. La luz se encendió a las 8:00 hrs. Y se apagó a las 20:00 hrs. Luego de una implantación crónica de electrodos para estudios electroencefalográficos (EEG) y electromiográficos (EMG), los registros fueron obtenidos mediante un sistema automatizado. Se consideraron 5 días de registro. El protocolo experimental consistió en dos días basales; el día previo al comienzo de los paradigmas y el día posterior a estos. Los tres restantes consistieron en privaciones de 20, 40 y 60 minutos, las cuales fueron alternadas en su orden por rata. Los experimentos demostraron un mayor número de intentos por entrar a REM durante el periodo de cada paradigma experimental. Por otro lado también se encontró un aumento en el tiempo en la cantidad de REM durante el periodo de recuperación de cada rata (rebote compensatorio) y este efecto fue mucho más marcado en el paradigma de 60 minutos, sobre todo al pasar aproximadamente una hora desde el inicio de la recuperación. Además de esto, los episodios de NREM durante el periodo de recuperación fueron mucho más propensos a pasar a REM que a Vigilia. En síntesis los efectos que causa una privación de mucho menor duración en relación a las privaciones de largo plazo, son muy similares entre sí, y además se manifiesta la homeostasis de corto plazo del sueño REM a este nivel de privación

<p>Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.</p><p>T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis. </p><p>In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies. </p><p>In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.</p><p>The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.</p> / Dissertation

Malgré son potentiel, l’hétérosis a rarement été étudié, et encore moins exploité, chez les levures, espèces d’intérêt biotechnologique majeur. Ce travail avait pour objectif d’explorer ce phénomène chez deux espèces de levure, Saccharomyces cerevisiae et S. uvarum, dans des conditions proches de celles de l’œnologie. Pour la première fois des hybrides interspécifiques ont été inclus dans un dispositif diallèle complet. Un autre aspect original de ce travail résidait dans l’approche intégrative choisie, qui combinait l’étude de phénotypes aux niveaux métabolique, cellulaire et populationnel. Un panel de 66 souches (55 hybrides et leurs 11 parents) a été analysé pour 35 caractères à deux températures et avec trois réplicats, soit au total 396 fermentations alcooliques. Ces données nombreuses et complexes nous ont conduits non seulement à utiliser, mais aussi à développer divers outils statistiques et de modélisation originaux pour l’interprétation des données. Après avoir vérifié que les interactions nucléo-cytoplasmiques n’influençaient pas la variation des caractères étudiés, nous avons tout d’abord montré que les sources de variation (effet souche, effet température et interactions souche*température) différaient selon les types de caractères. Nous avons ensuite comparé globalement les trois groupes d’hybrides : intraspécifiques S. cerevisiae*S. cerevisiae, intraspécifiques S. uvarum*S. uvarum et interspécifiques S. cerevisiae*S. uvarum, et avons observé que l’hybridation interspécifique pouvait engendrer des phénotypes présentant de meilleures aptitudes œnologiques et une homéostasie supérieure à celle des hybrides intraspécifiques. Ce dernier résultat pourrait expliquer que l’hybridation interspécifique soit si fréquente chez les levures naturelles et domestiquées. / Despite its biotechnological interest, heterosis has not commonly been studied or exploited in the yeast genus. This work aimed to explore this phenomenon within two yeast species well adapted to oenological conditions, Saccharomyces cerevisiae and S. uvarum. Eleven parental strains and their 55 intra- and inter-specific hybrids were phenotyped under enological conditions, at two temperatures in three replicates. A total of 396 alcoholic fermentations were characterized in depth through 35 phenotypic traits with original statistical and modeling tools. We first showed that, depending on the types of trait - kinetics parameters, life-history traits, enological parameters and aromas -, the sources of variation (strain, temperature and strain*temperature effects) differed in a large extent. Then we compared globally three groups of hybrids and their parents at two growth temperatures: intraspecific hybrids S. cerevisiae*S. cerevisiae, intraspecific hybrids S. uvarum*S. uvarum and interspecific hybrids S. cerevisiae*S. uvarum. We found that hybridization could generate multi-trait phenotypes with improved oenological performances and better homeostasis with respect to temperature. These results could explain why interspecific hybridization is so common in natural and domesticated yeast, and open the way to applications for wine-making.

Copper and iron are trace elements that form an indispensable part of many proteins and are crucial for the well-being of all cells. At the same time, the intracellular levels of these metals require careful regulation, as excess or deficiency may be lethal. P1B-ATPases are key players in metal homeostasis. They belong to the superfamily of P-type ATPases, transmembrane proteins present in virtually all life forms, are responsible for solute translocation across biological membranes. The goal of this thesis is to improve our understanding of the structural and functional roles of P1B-ATPases in metal homeostasis by focusing on the host-microbe interaction. The thesis first describes the importance of Cu+ distribution in the outcome of host-microbe interaction. Copper is an important element in host-microbe interactions, acting both as a catalyst in enzymes and as a potential toxin. Cu+-ATPases drive cytoplasmic Cu+ efflux and protect bacteria against metal overload. Many pathogenic and symbiotic bacteria contain multiple Cu+-ATPase genes within particular genetic environments, suggesting alternative roles for each resulting protein. This hypothesis was tested by characterizing five homologous Cu+-ATPases present in the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to different phenotypes and abnormal nodule development in the alfalfa host. Distinct responses were detected in free-living S. meliloti mutant strains exposed to metal and redox stresses. Differential gene expression was detected under Cu+, oxygen or nitrosative stress. These observations suggest that CopA1a maintains the cytoplasmic Cu+ quota and its expression is controlled by Cu+ levels. CopA1b is also regulated by Cu+ concentrations and is required during symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are necessary for the assembly of two different cytochrome c oxidases at different stages of bacterial life. CopA3 is a phylogenetically distinct Cu+-ATPase that does not contribute to Cu+ tolerance. It is regulated by redox stress and required during symbiosis. We postulated a model where non-redundant homologous Cu+-ATPases, operating under distinct regulation, transport Cu+ to different target proteins. In its second part, the thesis describes the novel Fe2+-ATPases and their influence in the host-microbe interaction. Little is known about iron efflux transporters in bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P1B4-ATPase, in cytoplasmic Fe2+ efflux has been proposed. We report here the distinct roles of mycobacterial P1B4-ATPases in the homeostasis of Co2+ and Fe2+. Mutation of Mycobacterium smegmatis ctpJ affects the homeostasis of both ions. Alternatively, a M. tuberculosis ctpJ mutant is more sensitive to Co2+ than Fe2+, while mutation of the homologous M. tuberculosis ctpD leads to Fe2+ sensitivity but no alterations in Co2+ homeostasis. In vitro, the three enzymes are activated by both Fe2+ and Co2+ and bind one equivalent of either ion at their transport site. However, equilibrium binding affinities and activity kinetics show that M. tuberculosis CtpD has higher affinity for Fe2+ and twice the Fe2+ stimulated activity than the CtpJs. These parameters are accompanied by a lower activation by and affinity for Co2+. Analysis of Fe2+ and Co2+ binding to CtpD by X-ray spectroscopy shows that both ions are coordinated by 5-6 O/N atoms with similar geometry. Mutagenesis studies suggest the involvement of invariant Ser, His and Glu in metal coordination. Interestingly, replacement of Cys in the conserved CPS sequence at the metal binding pocket leads to a large reduction in Fe2+ but not Co2+ binding affinity. We propose that CtpJ ATPases participate in the control of steady state Fe2+ levels. CtpD, required for M. tuberculosis virulence, is a high affinity Fe2+ transporter involved in the rapid response to iron dyshomeostasis generated upon redox stress. These studies provide significant insights into the metal selectivity, regulation, transport kinetics and functional diversity of homologous P1B-ATPases in Cu+ and Fe2+ homeostasis. Moreover, these biochemical characterizations can be integrated with the structural-functional analysis to elucidate the complex metal distribution networks.

Various types of mammalian cells exhibit the remarkable ability to adapt to external applied mechanical stresses and strains. This ability allows cells to maintain a stable endogenous mechanical tension at a preferred (homeostatic) level, which is of great importance for normal physiological function of cells and tissues, and for a protection from various diseases, including atherosclerosis and cancer. Previous studies have shown that the cell ability to maintain tensional homeostasis is cell type-dependent. For example, isolated endothelial cell cannot maintain tensional homeostasis, whereas clusters of endothelial cells can, more so the greater the size of the cluster is. On the other hand, cell clustering does not affect tensional homeostasis of fibroblasts and vascular smooth muscle cells. Underlying mechanisms for these behaviors of different cell types are largely unknown. In this study, we combined theoretical analysis and mathematical modeling to investigate several biophysical factors, including cluster shape and size, magnitude and dynamics of cellular traction forces, and applied shear forces that may influence tensional homeostasis in cells and clusters. We developed two-dimensional models of cells clusters of different shapes and sizes. To simulate temporal fluctuations of cell-extracellular matrix traction forces, we used a Monte Carlo approach. We also applied physical forces obtained from previous experimental measurements to the models. Results of the analysis and modeling revealed that cluster size, magnitude and dynamics of focal adhesion traction forces have a major influence on traction field variability, whereas the influence of cluster shape appears to be minor. The dynamics of traction forces seems to be related to cell types and it can explain why in certain cell types, such as endothelial cells, cell clustering promotes tensional homeostasis, whereas in other cell types, such as fibroblasts, clustering has virtually no effect on homeostasis. To further investigate mechanisms that may affect tensional homeostasis, we investigated the effect of applied steady shear stress on the traction field dynamics of endothelial cells and clusters. We applied steady shear stress to our two-dimensional model of cell clusters and then computed ensuing changes in the traction force variability. These simulations mimicked the effect of flow-induced shear stress on tensional homeostasis of endothelial cells and clusters. We found that under steady shear stress, temporal fluctuations of the traction field of endothelial cells became attenuated. This result agrees with the viewpoint that steady shear flow promotes tensional homeostasis in the endothelium. Together, results of this study advance our understanding of biophysical mechanisms that contribute to the cell ability to maintain tensional homeostasis. Furthermore, these results will help us to modify our current experimental procedures, as well as to design new experiments for our investigation of tensional homeostasis. / 2020-10-22T00:00:00Z

Metals are used as cofactors for enzymes, but are toxic in excess. In order to avoid the deleterious effects posed by metals, the cell must employ strict metal homeostasis systems. One such system is the Cue copper-resistance system in Salmonella enterica serovar Typhimurium (S. Typhimurium) which includes the periplasmic copper binding protein CueP. Previous studies have shown CueP to be a major periplasmic copper-sequestering protein that has a role in supplying copper to, and thus activating, the periplasmic Cu,Zn-superoxide dismutase enzyme SodCII (Osman et al., 2013). SodCII protects the cell from reactive oxygen species (ROS), due for example to the actions of the respiratory burst oxidase in host macrophages. However, despite its ability to sequester copper and activate SodCII, the precise physiological role of CueP in S. Typhimurium has remained unresolved since cueP mutants of S. Typhimurium strain SL1344 (the wild-type stain used in this study) do not exhibit a phenotype with respect to tolerance to copper or reactive oxygen species. In addition, the copper-binding mechanism of CueP and its interactions with other copper-binding proteins, including SodCII, have not been examined. An aim of this study was to establish a phenotype for a cueP mutant of S. Typhimurium with respect to copper and/or ROS tolerance. It was hypothesised that the possession of KatG (catalase) and multiple superoxide dismutases (SodCI, SodA and SodB), in addition to SodCII, by S. Typhimurium may confer functional redundancy with respect to copper and ROS tolerance. Hence mutants lacking katG (ΔkatG) or the various superoxide dismutase encoding genes (ΔsodA/ΔsodB/ΔsodCI/ΔsodCII) with and without functional cueP were generated. The ΔkatG mutants exhibited reduced catalase activity and reduced tolerance to hydrogen peroxide, consistent with the loss of KatG, however the additional loss of cueP did not reduce tolerance to hydrogen peroxide further. Similarly, tolerance to copper and extracellular superoxide was also unaltered in the ΔkatG/ΔcueP mutant. The tolerance of the various superoxide dismutase mutants to copper and various ROS was also unaffected by the presence or absence of CueP. To examine the role of CueP in SodCII activation in vivo, SodCII was over-expressed in S. Typhimurium (in a ΔsodA/ΔsodB/ΔsodCI/ΔsodCII background) with and without functional cueP and superoxide dismutase activity measured in both whole cells and periplasmic extracts. SodCII-dependent superoxide dismutase activity was successfully identified within the periplasmic extracts. However, surprisingly, the level of activity was unaffected by the presence 16 or absence of CueP and/or the addition of copper. It is possible that SodCII is thus able to scavenge sufficient copper for activity from the reagents used in these assays. Similarly, in an alternative approach to examine the role of CueP in vitro, both SodCII and CueP (WT and potential metal-binding residue mutant forms) were successfully over-expressed in E. coli and methods for their purification optimised (without the use of affinity tags). ICP-MS analysis indicated that a CuePC104S mutant contains > 18-fold less copper than the CueP WT protein. Furthermore, superoxide dismutase activity assays using purified proteins, indicated that the CuePC104S mutant was less able to activate SodCII than the WT CueP. Taken together, these results are consistent with a role for the Cys104 residue in copper-binding by CueP. Bioinformatics results suggest the presence of CueP or homologous genes in the presence of other bacteria, including pathogens such as Klebsiella, Yersinia and Shigella spp. Further understanding of the role of CueP and the systems used by S. Typhimurium to avoid both copper and ROS stress may inform the development of novel treatment strategies for bacterial diseases.

Submitted by Natalia Vieira (natalia.vieira@famerp.br) on 2016-05-19T22:20:23Z No. of bitstreams: 1 rosangelaakemihoshi_tese.pdf: 2556179 bytes, checksum: a5e900121bd799f888f7c902c2e959c4 (MD5) / Made available in DSpace on 2016-05-19T22:20:23Z (GMT). No. of bitstreams: 1 rosangelaakemihoshi_tese.pdf: 2556179 bytes, checksum: a5e900121bd799f888f7c902c2e959c4 (MD5) Previous issue date: 2015-09-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: Exercises promote changes in the Autonomic Nervous System, the mainly responsible for neurodegenerative functions. Due to its importance, several methods are used in evaluation such as Heart Rate Variability (HRV) analysis, considered an indirect technique to assess autonomic functions, but its use is still questioned. Objectives: To analyze the correlation between HRV indices and concentration of epinephrine and norepinephrine, and observe post-exercise recovery, by HRV linear and nonlinear indices and cardiopulmonary parameters. Methods: 28 participants, divided into two groups (N = 14) had their recovery after exercise evaluated. One group performed submaximal effort (55% V̇O2peak), for 5 minutes, and the other, supramaximal (110% of peak V̇O2) until exhaustion. During 90 minutes post-exercise, blood samples were collected, cardiopulmonary variables were registered and HRV was analyzed. Results: Between baseline and immediately postexercise, we detected that adrenaline and noradrenaline showed significant nonlinear correlation with RR intervals, heart rate, HRV linear indices and nonlinear parameters related to complexity and fractality. After exercise performed at 55% of peak V̇O2, recovery was observed between 30 and 60 minutes, whereas for exercise at 110%, 90 minutes were not enough for returning of variables to baseline levels. Conclusions: Plasma catecholamine concentrations after exercise presented correlation with HRV linear indices and nonlinear parameters related to complex and fractal characteristics of heartbeats, especially the Lyapunov exponent. The variables recovery occurred at different times, but the changes caused by supramaximal exercise were more expressive and lasting. / Introdução: A realização de exercícios físicos promove alterações no sistema nervoso autônomo, principal responsável pelas funções neurovegetativas. Devido à sua importância, diversos métodos são utilizados para avaliação, como a análise da Variabilidade da Frequência Cardíaca (VFC), considerada uma técnica indireta de acesso ao funcionamento autonômico, porém seu emprego é ainda questionado. Objetivos: analisar a correlação entre índices de VFC e concentração de adrenalina e noradrenalina, e observar a recuperação pós-exercício, a partir dos índices lineares e não lineares de VFC e de parâmetros cardiopulmonares. Métodos: 28 participantes, divididos em dois grupos (N=14), foram submetidos à avaliação da recuperação após exercício. Um grupo realizou esforço submáximo (55% do V̇O2pico), por 5 minutos, e o outro, supramáximo ( 110% do V̇O2pico) até a exaustão. Durante 90 minutos pós-exercício foram realizadas coletas sanguíneas, registros das variáveis cardiopulmonares e análise da VFC. Resultados: No período entre o basal e imediatamente após o exercício, detectou-se que adrenalina e noradrenalina apresentaram correlações não lineares significantes com intervalos R-R, frequência cardíaca, índices lineares de VFC e parâmetros não lineares relacionados à complexidade e fractalidade. Após exercício realizado a 55% do V̇O2pico, a recuperação foi verificada entre 30 e 60 minutos, enquanto que para o esforço a 110%, 90 minutos não foram suficientes para o retorno das variáveis aos níveis basais. Conclusões: Concentrações de catecolaminas plasmáticas após exercícios apresentam correlação com índices lineares de VFC e parâmetros não lineares relacionados às características complexas e fractais dos batimentos cardíacos, sobretudo o expoente de Lyapunov. A recuperação das variáveis analisadas ocorreu em tempos diferentes, porém as alterações promovidas pelo exercício supramáximo foram muito mais expressivas e duradouras.

Metabolic activity is an important functional parameter of a living cell. Microscopic techniques are demanded to resolve the heterogeneity of metabolic activity from cell to cell and among subcellular compartments. Towards this, work in the Min lab has been dedicated to developing a prevailing metabolic imaging platform that couples chemical imaging by stimulated Raman scattering (SRS) microscopy with small vibrational tags on precursor molecules. This thesis describes efforts along metabolic imaging by SRS microscopy, with focus on visualizing protein and lipid homeostasis. Chapter 1 describes the design principle of metabolic imaging, including selection of vibrational tags and setup of SRS microscopy, and an overview of successful demonstrations of metabolic imaging in protein and lipid metabolism. Chapter 2 describes adoption of such principle to visualize protein turnover with 13C-phenylalanine metabolic labeling under steady-state condition and various perturbations. The rest of this thesis (Chapters 3-6) switches focus to fatty acid metabolism and cellular lipid homeostasis. As the minimal tagging in vibrational imaging preserves the physicochemical property of lipid molecules to the largest extent, it motivated me to revisit fatty acid metabolism from a biophysical perspective. Bearing the question in mind whether the non-equilibrium metabolic activity could drive phase separation in biological membranes, I thus look into the principle of membrane organization and its implication in biological membranes in Chapter 3. Then in Chapter 4, I describe the discovery and characterization of previously unknown phase separation in endoplasmic reticulum (ER) membrane caused by lipid synthesis. In this case, metabolic imaging by SRS enables identification of solid-like domains formed by saturated fatty acid (SFA) metabolites. This observation further raises the question whether phase separation bears any functional roles in the adverse effects of SFAs (or lipotoxicity). Towards this, Chapter 5 introduces the background of lipotoxicity including its definition and models. Then I review proposed mechanisms for lipotoxicity, which point to the central role of ER in mediating the stress transduction. In Chapter 6, I present our findings that suggest the association of the observed solid-like domains with ER structural remodeling and local autophagic arrest. Together, these efforts demonstrate the valuable capability of SRS imaging to reveal metabolic heterogeneity and how this aids in the investigation of metabolic stress.

Includes bibliographical references (leaves 169-184) / xiv, 184 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the concept of membrane homeostatis, which implies that biological membranes tend to maintain a constant level of lipid fluidity in the face of potential exogenous and endogenous pertubations. Manipulations of dietary cholesterol and/or saturated (coconut oil) v/s unsaturated (sunflower seed oil) fatty acids have been used to study the relationship between membrane lipid composition, membrane lipid fluidity and membrane-bound enzymes of lipid metabolism; and, to see whether these enzymes act co-ordinately for the maintenance of a membrane homeostatis under these dietary conditions. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1986

Abstract Gibberellins (GAs) are a class of plant hormones that regulate many aspects of plant growth and development including seed germination, stem elongation and fruit development. To investigate the regulation of GA biosynthesis and the impact of altered GA levels on plant growth and development, transgenic pea (Pisum sativum L. cv. Carneval) plants were generated to overexpress PsGA3ox1 (codes for GA 3-hydroxylase which converts GA20 to bioactive GA1) under the control of the CaMV-35S promoter. Increased expression of the transgene PsGA3ox1 was correlated with altered plant phenotype including longer internodes, larger stipules and tendrils, and longer pods. Transgenic lines also showed upregulation of the GA catabolic genes PsGA2ox1 and/or PsGA2ox2, suggesting that GA1 substrate-induced feedback regulation also occurs to maintain GA homeostasis. Changes in endogenous GAs, quantified using an isotope dilution method, indicated that an increased flux in GA biosynthesis occurred in the expanding internodes, stipules and tendrils of the PsGA3ox1-overexpressor lines. Higher bioactive GA1 levels and growth were correlated with lower PsGA2ox1 transcript levels in elongating internodes, and oscillation of these parameters between adjacent elongating internodes in the PsGA3ox1-overexpression lines suggests that coordination of bioactive GA levels and growth occurs between adjacent internodes. During germination and early seedling growth, GA gene expression studies suggested that PsGA3ox1-overexpression increased the flux through to bioactive GA in the cotyledons, shoots and roots of pea seedlings, resulting in longer shoots but shorter roots. Auxins are a class of plant hormones involved in growth and differentiation of plants that can influence GA biosynthesis and action. The location and action of auxins is in part regulated by auxin carrier proteins. The expression patterns of the putative auxin efflux carrier genes PsPIN1 and PsPIN2 in elongating internodes were correlated with vascular re-patterning events in this tissue, and PsGA3ox1-overexpression appears to increase internode PsPIN1 and PsPIN2 transcript abundance and the formation of the vascular connections between the internode and the axillary buds. Overall, characterization of PsGA3ox1-overexpressor lines in pea demonstrated that bioactive GA levels are tightly regulated in pea tissues for the coordination of plant growth and development. / Plant Science