<p>Amino acids are key nutritional stimuli that are both substrate for muscle protein synthesis (MPS), and signaling molecules that regulate the translational machinery. There is a dose-dependent relationship between protein intake and MPS that differs between young and elderly subjects. The current thesis contains results from three separate studies that were conducted to examine to potential to enhance smaller doses of protein, known to be suboptimal in their capacity to stimulate MPS, through supplementation with specific amino acids, namely leucine and citrulline.</p> <p>The first two studies examined the potential to enhance the muscle protein synthetic capacity of a smaller, suboptimal dose of whey protein with leucine. In study one, we concluded that leucine supplementation of a suboptimal dose of protein could render it as effective at enhancing rates of MPS as ~four times as much protein (25 g) under resting conditions, but not following resistance exercise. In study two, we examined the potential of leucine and branched-chain amino acids to enhance the MPS response of a suboptimal dose of protein within the context of mixed macronutrient ingestion. We concluded that supplementation with a relatively high dose of leucine could render it as effective at enhancing MPS rates as ~four times as much protein (25 g) under both resting and post-exercise conditions.</p> <p>In study three, we examined the potential of citrulline supplementation to enhance blood flow, microvascular circulation, and MPS in response to a suboptimal dose of whey protein in elderly subjects. We concluded that supplementation of a suboptimal dose of protein with citrulline did not augment bulk blood flow or muscle microvascular circulation. The major findings from the works presented in this thesis is that smaller doses of protein that normally elicit a suboptimal increase in MPS can be made more anabolic when supplemented with specific amino acids.</p> / Doctor of Philosophy (PhD)

This work presents the application of spiroligomers as inhibitors of protein-protein interactions. After the discovery of an acyl-transfer coupling reaction by Dr. Zachary Brown, a previous graduate student of Schafmeister group, the synthesis of highly functionalized spiroligomers that mimic the helical domain of p53 was undertaken before each molecule was tested for binding to HDM2, a natural binding partner of p53. A library of molecules was synthesized on solid support that altered the stereochemistry along the spiroligomer as well as the presented functional groups. It was determined that spiroligomers enter human liver cancer cells through passive diffusion and induces a biological response in both a dose- and time-dependent manner. The synthesis of additional spiroligomer analogues achieved low micromolar to high nanomolar range activity during screening in direct and competitive binding assays. In parallel to the project above, a series of spiroligomers that mimic the side chains of the leucine zipper region of Max were synthesized in an effort to disrupt the interaction of the protein with c-Myc. The series of compounds contained various stereocenter combinations and different functional groups as before but were made in solution before testing for inhibition. Initial binding assays resulted in low micromolar activity, however, secondary assays (ELISA and cellular assays) did not confirm the inhibitory effect of spiroligomers on the c-Myc/Max heterodimer. In summary, this work illustrates that spiroligomers are capable mimics of helical peptides and can induce a biological response. / Chemistry

Soybean mosaic virus (SMV), has been the cause of numerous and often devastating disease epidemics, causing reduction in both the quality and quantity of soybeans worldwide. Two important genes for resistance to SMV are Rsv1 and Rsv4. Alleles at the Rsv1 locus have been shown to control resistance to all but the most virulent strain of SMV. This locus has been mapped previously to the soybean F linkage group. Rsv4 is an SMV resistance locus independent of Rsv1 and confers resistance to all strains of SMV. This locus has not been mapped previously. The purpose of this study is to investigate the two genomic regions that contain these vitally important resistance genes. A population of 281 F2 individuals that had previously been genotyped for reaction to SMV was evaluated in a mapping study which combined bulk segregant analysis with Amplified Fragment Length Polymorphism (AFLP). A Rsv4-linked marker, R4-1, was identified that mapped to soybean linkage group D1b using a reference mapping population. More than 40 markers were mapped in the Rsv4 segregating population including eleven markers surrounding Rsv4. This will provide the necessary framework for the fine mapping of this important genetic locus. Previous work has located Rsv1 to a genomic region containing several important resistance genes including Rps3, Rpg1, and Rpv. An RFLP probe, NBS5, whose sequence closely resembles that of several cloned plant disease resistance genes has been mapped to this chromosomal region. The efficacy of using this sequence to identify potential disease resistance genes was assessed by screening a cDNA library to uncover a candidate disease resistance gene which corresponds to this NBS5 sequence. Two related sequence classes were identified that correspond to NBS5. Interestingly, one class corresponds to a full length gene closely resembling other previously cloned disease resistance genes offering evidence that this NBS5-derived clone is a candidate disease resistance gene. A new marker technique was developed by combining the speed and efficiency of AFLP with DNA sequence information from cloned disease resistance genes. Using this strategy, three new markers tightly linked to Rsv1 were identified. One of these markers, which maps 0.6 cM away from Rsv1, has motifs consistent with other cloned disease resistance genes, providing evidence that this approach is an efficient method for targeting genomic regions where disease resistance genes are located. / Ph. D.

Branched-chain amino acids (BCAA) are a group of essential amino acids consisting of leucine, isoleucine, and valine. Leucine, in particular, has signaling functions affecting protein and energy metabolism. Plasma leucine concentration is positively correlated with obesity and associated metabolic disorders. We set out to test the hypothesis that metabolic dysfunction from high fat diets precedes dysfunctional BCAA metabolism. First, BCAA were supplemented to neonatal pigs for 4 weeks to evaluate whether the anabolic signaling function of leucine could increase muscle growth when fed for a longer duration than in previous studies. Neither normal pigs nor low birth weight pigs, which have naturally impaired muscle growth, grew better in response to BCAA supplementation, despite low birth weight pigs expressing less of the leucine sensing protein Sestrin2 in skeletal muscle. Furthermore, high plasma BCAA concentrations caused by the experimental diets had no effect on adiposity, liver fat accumulation, or expression of genes related to fatty acid synthesis, mitochondrial biogenesis, or energy expenditure in the pigs' livers. Having produced strong evidence that long term BCAA supplementation neither improves lean growth nor causes abnormal fat metabolism, we then tested whether fat supplementation changes BCAA metabolism. Pigs were fed milk replacer formula with either low energy (Control), or high energy from long-chain fatty acids (LCFA) or medium-chain fatty acids (MCFA) for 22 days. Although high fat diets did not increase plasma BCAA concentrations, the MCFA diet in particular caused metabolic changes which could lead to fatty liver disease and decreased oxidative BCAA disposal. Expression of fatty acid synthesizing genes were increased in the livers of pigs fed MCFA formula compared to Control and LCFA formula. Oxidation of α-ketoisocaproic acid was decreased in liver homogenate of pigs fed MCFA and LCFA formulas compared to Control. Additionally, hepatic oxidation of α-ketoisovalerate was decreased, and plasma concentration of α-ketoisovalerate was consequently increased, in pigs fed MCFA formula compared to Control, with LCFA formula causing intermediate results. In future research, it would be valuable to feed high MCFA formula for a longer period of time to determine whether nonalcoholic fatty liver disease will develop, and whether plasma BCAA concentrations will increase due to decreased oxidation. Overall, these studies concluded that long term BCAA supplementation does not increase muscle growth in neonatal pigs, but there is also no indication that they cause obesity or dysfunctional fat metabolism. On the other hand, high fat diets cause impairments in BCAA catabolism which may precede elevated plasma BCAA concentrations. / Doctor of Philosophy / Branched-chain amino acids (BCAA) are essential amino acids which are abundant in plant and animal proteins. In addition, the BCAA leucine has functions in protein and energy metabolism. Leucine consumption induces a signal to build new muscle protein. However, leucine concentration is also higher in blood plasma of obese individuals than in non-obese individuals, which has caused uncertainty regarding the safety of leucine consumption. In order to demonstrate that leucine does not cause obesity, we set out to test the hypothesis that high fat diets cause decreased breakdown of BCAA. In the first study, we tested whether one month of BCAA supplementation could increase muscle growth in neonatal pigs. Neither normal pigs nor low birth weight pigs, which have naturally impaired muscle growth, grew better in response to BCAA supplementation, despite low birth weight pigs expressing less of a leucine sensing protein in skeletal muscle. Furthermore, BCAA supplementation caused higher BCAA concentrations in blood plasma, but did not cause pigs to gain more fat, or cause any changes in liver fat metabolism. Having produced strong evidence that BCAA supplementation neither improves lean growth nor causes abnormal fat metabolism, we then tested whether fat supplementation changes BCAA metabolism. Pigs were fed milk replacer formula which was either low calorie (Control), or high calorie from animal fat, which is rich in long-chain fatty acids (LCFA) or high calorie from coconut oil, which is rich in medium-chain fatty acids (MCFA). Although high fat diets did not increase blood plasma BCAA concentrations, the MCFA formula in particular caused changes which could lead to fatty liver disease and decreased breakdown of BCAA. Genes which synthesize new fatty acids were increased in the livers of pigs fed MCFA formula compared to those fed LCFA and Control formulas. Furthermore, liver samples taken from pigs fed the MCFA and LCFA formulas were less able to fully break down metabolites of leucine compared to pigs fed the Control formula. In addition, liver samples from MCFA fed pigs were less able to fully break down metabolites of the BCAA valine, which led to higher concentrations of that metabolite in the blood plasma of pigs fed MCFA formula compared to pigs fed LCFA or Control formula. In the future, it would be valuable to feed a high MCFA formula for a longer period of time to determine whether nonalcoholic fatty liver disease will develop, and whether blood plasma BCAA concentrations will increase due to decreased breakdown. Overall, these studies concluded that long term BCAA supplementation does not increase muscle growth in neonatal pigs, but there is also no indication that they cause obesity or dysfunctional fat metabolism. On the other hand, high fat diets cause impairments in BCAA breakdown which may lead to elevated BCAA concentrations in blood plasma.

Livestock across the world contract several infectious diseases of both bacterial and viral origin. Swine brucellosis caused by Brucella suis and swine influenza caused by Influenza A virus affect both domestic and feral swine populations. Both the diseases have zoonotic potential to cause disease in humans with serious complications apart from inflicting huge economic losses. Infected feral swine can also act as a source of spread and outbreak where the disease is not endemic. At present, there is no vaccine available for swine brucellosis. The currently used swine influenza vaccine may not be effective against influenza strains like the recent H1N1 strain that caused a pandemic. To develop an effective bivalent vaccine for swine against these two diseases, a leucine auxotroph of the USDA approved vaccine B. abortus strain RB51 was constructed along with leuB gene complementing plasmid pNS4 to over-express antigens from Brucella and influenza. This antibiotic resistance free system over-expressed Brucella derived antigens SOD, L7/L12 and WboA in three different constructs. Against a virulent challenge of B. suis, the candidate vaccine strain over-expressing both SOD and WboA protected mice more significantly than the control group and was also found to be better protective than other candidate vaccine strains over-expressing either SOD and L7/L12 together or SOD alone. Immunoassays (ELISA) suggested that the protection afforded is Th1 type mediated immune response, as cytokine IFN-γ and IgG2a antibody sub-isotype was observed in the splenocyte culture supernatant and serum samples respectively. The strain RB51leuB platform was not expressing influenza derived antigens Hemagglutinin (HA) and Nucleoprotein (NP) when screened for expression by immunoblot. Influenza antigens, HA, NP and ectodomain of matrix protein M2e, were not found to be expressing even after optimizing their codon usage to suit Brucella tRNA preference. However, RT-PCR showed that the influenza genes mRNA were produced. In conclusion, this dissertation describes the construction of an environmentally safe antigen over-expression platform and successful employment of the system as a candidate vaccine in protecting mice against B. suis challenge. This new platform is a potential candidate for developing vaccines against other infectious diseases of livestock. This document also discusses alternate strategies for expressing influenza antigens in a Brucella platform. / Ph. D.

In the United States corn is one of the leading agricultural products and one of the top exports. The majority of U.S corn is grown in the Midwestern region of the U.S. known as the Corn Belt where the bacterial disease Stewart's Wilt reduces crop yield. Pantoea stewartii subsp. stewartii (Pss) is transmitted into corn via the corn flea beetle insect vector, Chaetocnema pulicaria. As the beetle feeds on the corn plant leaves, Pss deposited in beetle feces enter the leaf through lesions. The early stage of Pss infection begins in the mesophyll apoplast of the corn leaf where a type III secretion system (T3SS) and its associated effectors induce water soaking (WS) and nutrient release. Ultimately, Pss will enter the plant xylem apoplast (will be referred to as the xylem) and use quorum sensing (QS) to initiate a lifestyle shift. Within the xylem, Pss grows to high cell density and secretes exopolysaccharide (EPS), forming a biofilm which eventually obstructs water transport, leading to wilting and necrosis. Previous Tn-Seq experiments provided insights into genes that are essential for in planta survival, including the master transcriptional regulator, Leucine-responsive Regulatory Protein (Lrp). To better understand the role of Lrp when Pss inhabits the xylem, RNA-Seq experiments comparing Pss wild-type and ∆lrp strains grown in planta were conducted to ascertain differential gene expression. The RNA-Seq data was further analyzed using DESeq2 and validated using qRT-PCR methods. Following validation, the Pss genome was annotated using Blast2GO software and genes upregulated and downregulated by Lrp were linked with biological processes. Lrp was found to be involved in regulating capsule biosynthesis and nitrogen-associated assimilation and metabolism during Pss survival in the xylem. This provides further insight into how Pss contends with harmful host defense compounds and extracts scarce nutrients present in the in planta xylem environment. A corn xylem fluid extraction method was developed that has enabled more physiologically relevant growth experiments to be conducted in vitro. Extracted xylem fluid was used to grow Pss wild-type and ∆lrp mutant strains as monocultures to observe any differences in growth patterns in different growth media. When grown separately in xylem fluid or Luria-Bertani (LB) medium, the Pss wild-type and ∆lrp mutant strains grew at similar rates and to final cell densities . The Pss ∆lrp mutant strain greatly outcompeted the wild type when grown together in LB medium. However, when the two Pss strains were growth together in xylem fluid, a shift in relative competition was observed, providing evidence of the wild type slightly outcompeting the ∆lrp mutant. Analysis of the composition of extracted xylem fluid through metabolomics will help define the nutrients specifically utilized by Pss in planta. Altogether, the outcome of these research projects was to provide pertinent discoveries to contribute to understanding the mechanisms used by Pss to survive in the corn xylem environment. Broadly, increased understanding of Pss pathogenesis may translate to understanding pathogenesis mechanisms in other bacterial wilt-disease causing plant pathogens. / Master of Science / Corn is a significant agricultural product and export in the United States. This important crop is used as a food source for humans, a primary nutrient source of livestock, and a major ingredient for corn-based industries manufacturing commodities such as culinary additives, biofuels, and preservatives. Certain bacteria are greatly beneficial to plants, able to increase their overall health and growth, while other bacteria share a more insidious relationship with plants and cause disease. The research discussed in this thesis focuses on the bacterial pathogen Pantoea stewartii subspecies stewartii (Pss), the causal agent of Stewart's wilt disease in corn. Pss grows inside the plant xylem (vascular tissues which distribute water throughout the plant) and forms a biofilm that causes plant wilt leading to lower crop yield and even plant death. Previous research on Pss identified important genes for successful Pss survival inside the corn plant xylem. One of those genes codes for the Leucine-responsive Regulatory Protein (Lrp). Using a combination of experimental (RNA-Seq) and computational (bioinformatics) analyses, Lrp was found to control other genes related in biological process important for living inside the plant, necessary for the metabolism of available nutrients and production the protect slime layer within biofilm. By better understanding the key bacterial genes needed for Pss to grow inside the xylem, new disease intervention strategies can be developed to disrupt these genes and impede the ability of the bacterium to infect the plant. A second part of this research project was to develop a method for extracting corn xylem fluid from the plant. Using this extracted xylem fluid, experiments could be conducted in the laboratory to study Pss growth in more detail. The original strain of Pss (wild type) was grown separately and in combination with a Pss mutant lacking the Lrp gene in the extracted xylem fluid. Both strains grew similarly in the xylem fluid, but the wild type slightly outcompeted the mutant strain when they were grown in competition. Future work in the lab will use extracted xylem fluid to determine its precise nutrient composition and the development of synthetic xylem fluid that will enable a more detailed analysis of mechanisms used by Pss to grow in the xylem. Work on Pss serves as a model for the study of other bacterial wilt-disease causing pathogens.

Les lésions hépatiques observées au cours de l'obésité (NAFLD, stéatopathie non alcoolique) s'étendent de la stéatose isolée à la stéatohépatite (NASH, stéatohépatitie non alcoolique), la fibrose, la cirrhose et au carcinome hépatocellulaire. L'identification des mécanismes de recrutement des cellules immunitaires par le foie stéatosique est une étape clé dans la compréhension du déclenchement de l'inflammation hépatique et la recherche de nouvelles cibles thérapeutiques. Au cours de l’obésité, la stéatose sensibilise le foie au lipopolysaccharide (LPS), qui active la voie pro-inflammatoire NFκB. Nous avons récemment montré que: 1) la stéatose induisait une augmentation du recrutement lymphocytaire (TCD4+, TCD8+ et B) vers le foie mais également une augmentation de la réponse des lymphocytes TCD4+ à la chimiokine CXCL12 (SDF-1α), dont le récepteur est CXCR4 ; 2) GILZ (Glucocorticoid-Induced Leucine Zipper), une protéine induite par les glucocorticoïdes (GCs), inhibait la voie NFkB et jouait un rôle clé dans l’inflammation hépatique au cours de la consommation excessive d’alcool.Le but de ce travail était d’étudier les mécanismes de l’inflammation hépatique liée à l’obésité. Au cours de mon travail, nous avons montré que le chimiotactisme des lymphocytes TCD4+ à la chimiokine CXCL12 était augmenté non seulement chez les souris obèses mais également chez des patients ayant une NASH. L’augmentation de l’effet chimiotactique de CXCL12 était due à une augmentation de l’affinité de CXCL12 à son récepteur CXCR4. La migration anormale des lymphocytes T CD4+ vers le foie stéatosique était réversible pharmacologiquement en inhibant la liaison de CXCL12 à CXCR4 par AMD3100 (antagoniste deCXCR4). Le déficit d’expression et l’altération de l’induction du facteur anti-inflammatoire GILZ dans les cellules des Kupffer des souris obèses étaient responsables de la sensibilisation de ces cellules au LPS. Cette altération était liée à la diminution de l’expression du récepteur aux glucocorticoïdes (GR) dans les cellules de Kupffer des souris obèses. La surexpression de GILZ dans l’obésité en utilisant des souris trangéniques restaurait la tolérance hépatique au LPS. Ces anomalies des lymphocytes TCD4+ et de l’expression de GILZ dans les cellules de Kupffer participent au déclenchement d’une inflammation hépatique sur un foie stéatosique et pourraient représenter de nouvelles cibles thérapeutiques / Non alcoholic fatty liver disease (NAFLD) includes a spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The identification of the mechanisms involved in the recruitment of immunity cells by the fatty liver is a key in the comprehension of the onset of liver and the finding of new therapeutic targets. In obesity, steatosis sensitizes the liver to the lipopolysaccharide (LPS) from the gastrointestinal tract and the NFkB pro-inflammatory pathway is activated. We recently showed that: 1) the steatosis led to an increase recruitment of lymphocytes (TCD4+, TCD8+ and B) by the liver but also an hyperresponsive of CD4+T cells to CXCL12 (SDF-1"), the ligand of CXCR4; 2) GILZ(Glucocorticoid-Induced Leucine Zipper), a protein induced by glucocorticoids (GCs), inhibits the nuclear factor kB pathway and plays a key role in alcoholic hepatitis.This aim of my work was to study the mechanisms involved in obesity-related liver inflammation.I demonstrated that the chemotaxis of CD4+T cells to CXCL12 was increased not only in obese mice but also in patients with NASH. This increased chemotactisme of CXCL12 was due to an increase of the affinity ofCXCL12 to its receptor. The abnormal migration of CD4+T lymphocytes to the fatty liver was reversible by pharmacologically inhibiting the binding of CXCL12 to CXCR4 using AMD3100.The decreased expression and the impairment of the induction of the anti-inflammatory factor GILZ in Kupffer cells from obese mice was responsible for a sensitization of these cells to LPS. This impairment was due to a decrease of the glucocorticoid receptor (GR) expression in Kupffer cells from obese mice. The overexpression of GILZ level in obese transgenic mice restored the liver tolerance to LPS. These abnormalities of CD4+T lymphocytes and the GILZ expression in Kupffer cells contribute to the onset of liver inflammation in obesity and may represent new therapeutic targets.

Les racines ont deux grands rôles. Le premier est le prélèvement de l’eau et des éléments nutritifs et le second est l’ancrage dans le sol. Identifier les gènes responsables de la mise en place des tissus et de l'architecture du système racinaire est donc essentiel pour pouvoir améliorer les variétés de riz soumises à des stress abiotiques de plus en plus fréquents et nombreux du fait du changement climatique. Au cours de cette thèse, j'ai réalisé une analyse fonctionnelle du gène DEFECTIVE IN OUTER CELL LAYER SPECIFICATION (DOCS1) qui appartient à la famille des récepteurs kinases à répétitions riches en leucine (LRR-RLK). Ces protéines sont composées de deux domaines principaux: un domaine extra-cytoplasmique composé de répétitions LRR et un domaine kinase intra-cytoplasmique. Un mutant de ce gène, nommé c68, possède une mutation non-sens dans le domaine kinase. Les plantes mutantes c68 présentent plusieurs phénotypes: une sensibilité accrue à l'aluminium, une réduction du nombre et de la taille des poils absorbants dans les racines, et des couches d’exoderme/épiderme d’identité mêlée. Le premier chapitre de la thèse porte sur l’étude conjointe de lignées knock-out CRISPRs du gène DOCS1 et de c68. Nos résultats ont montré que les mutants c68 et CRISPRs présentaient les mêmes phénotypes : sensibilité à l’aluminium, défauts des poils absorbants et tissus externes d’identité mixte. Ces résultats suggéraient que chez le mutant c68, soit la protéine DOCS1 n'était pas fonctionnelle, soit elle n'était pas traduite. Nos analyses phénotypiques ont aussi révélé que tous les mutants présentaient des défauts de réponse à la gravité à différents stades de développement. A 3 jours, un retard de réponse à la gravité était observé pendant la première heure après gravistimulation. Les plantules mutantes présentaient aussi des défauts de localisation d’un transporteur d’auxine. A 40 jours, nous avons observé que l'angle du cône racinaire des plantes mutantes était plus ouvert que celui des plantes sauvages. Deux gènes liés à l’auxine et plusieurs QTLs ont déjà été identifiés comme participant à ce phénotype chez le riz. Dans la suite de notre étude, nous avons donc cherché à identifier de nouveaux QTLs et gènes impliqués dans ce phénotype morphologique par étude d'association pan-génomique dans deux panels Indica et Japonica. Toutes les accessions de l'écotype bulu d'Indonésie et trois japonicas tempérés d'Asie du Sud présentaient un angle du cône racinaire très ouvert. En utilisant un modèle mixte associé à une technique de ré-échantillonnage, 55 QTLs ont été détectés. L'analyse des gènes sous-jacents ou voisin (+/- 50kb) a identifié 539 gènes, dont 6 LRR-RLK, 5 gènes liés à l’auxine et 5 gènes avec une fonction validée dans le développement ou l'architecture racinaire. Une approche complémentaire par cartographie génétique classique est proposée pour identifier les gènes en cause dans la ou les mutations à angle du cône racinaire très ouvert. Des perspectives de poursuite du travail effectué sont aussi présentées afin de déterminer si le phénotype affectant l'angle du cône racinaire induit par les mutations du gène DOCS1 ou des nouveaux gènes identifiés est lié à des perturbations des flux d’auxine. / Roots have two major roles. The first one is to uptake water and nutrients and the second one is to anchor plants into the ground. Identifying the genes responsible for the establishment of tissues and architecture of the root system is essential to improve rice varieties subject to increasingly frequent and numerous abiotic stresses due to climate change. During my PhD, I undertook a functional analysis of the DEFECTIVE IN OUTER CELL LAYER SPECIFICATION (DOCS1) gene which belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family. These proteins are composed of two main domains: an extra-cytoplasmic domain containing LRR repeats and a cytoplasmic kinase domain. A mutant of this gene, named c68, carries a nonsense mutation in the kinase domain. The c68 mutant plants show several phenotypes: increased sensitivity to aluminum, reduced number and size of root hairs, and layers of external tissues with exodermis/epidermis mixed identity. The first chapter of the thesis focuses on the joint study of knockout CRISPRs lines of the DOCS1 gene and c68. Our results showed that the c68 and CRISPRs mutants displayed the same phenotypes: sensitivity to aluminum, defects in root hairs and mixed identity of external tissues. These results suggested that in the c68 mutant, either the DOCS1 protein was not functional, or the protein was not translated. Our phenotypic analyses also showed that all mutants exhibited impaired gravity responses at different development stages. At 3 days, a delay of response to gravity was observed during the first hour after gravistimulation. Mutant seedlings also had defects in an auxin transporter localization. At 40 days, we observed that the root cone angle of mutant plants was more open than that of wild-type plants. Two genes associated with auxin and several QTLs have been identified as contributing to this phenotype in rice. In the rest of our study, we therefore tried to identify new QTLs and genes involved in this morphological phenotype by a genome-wide association study in two Indica and Japonica panels. All accessions of the bulu ecotype from Indonesia and three South Asian temperate japonica had a very open root cone angle. Using a mixed model associated with a resampling technique, 55 QTLs were detected. The analysis of the underlying or neighbor (+/- 50kb) genes identified 539 genes, including 6 LRR-RLK, 5 genes related to auxin and 5 genes with a function validated in root development or architecture. A complementary approach by classical genetic mapping is proposed to identify genes involved in the mutation(s) involved in very open root cone angle. Prospective research lines are also presented to determine if the root cone angle phenotype , induced by DOCS1 or by newly identified genes, is linked with disruption of auxin fluxes.

Doctor of Philosophy / Department of Plant Pathology / Richard B. Todd / Fungi can be found living in a range of environments, including soil and the ocean, and as pathogens of plants and animals. The ability of fungi to adapt to diverse and changing environments is dependent on their ability to sense and respond to an array of signals, including the presence or absence of nitrogen nutrients. Fungi can utilize a diverse array of nitrogen nutrients and do so in a regulated and preferential manner. When preferred nitrogen nutrients such as ammonium and glutamine are present (nitrogen sufficiency), genes required for the utilization of alternative nitrogen sources are not expressed. In the absence of a preferred nitrogen source (nitrogen limitation) the genes for utilization of alternative nitrogen sources are transcriptionally derepressed and can be induced by the presence of a particular nitrogen nutrient, such as nitrate or proline. In the absence of any nitrogen nutrient (nitrogen starvation) the expression of some genes is further elevated. In filamentous fungi the expression of genes required for the utilization of nitrogen nutrients is coordinated by the orthologs of the conserved Aspergillus nidulans GATA transcription factor AreA, which activates transcription of nitrogen utilization genes. AreA activity is controlled by autogenous transcriptional activation, mRNA transcript stability, regulated nucleo-cytoplasmic distribution, and interactions with NmrA, AreB and TamA. The combined effect of these regulatory mechanisms generally results in AreA being inactive during nitrogen sufficiency and active during nitrogen limitation and nitrogen starvation. However, during nitrogen sufficiency AreA remains active at the promoters of some genes, including gdhA, which encodes the key nitrogen assimilation enzyme NADP-dependent glutamate dehydrogenase. In this work we have used both classical genetics and next generation sequencing approaches to examine regulated gene expression and how AreA activity is modulated, primarily during nitrogen sufficiency. We have studied regulation of gdhA to characterize how AreA evades nitrogen metabolite repression. We identify leucine biosynthesis as being a key regulatory signal involved in gdhA expression and characterize the genes required for leucine biosynthesis. We also show that TamA regulates the gdhA promoter by direct DNA binding, which requires interaction with AreA. We have also characterized repression of AreA to identify a potential mode of NmrA corepressor action. Finally, we have characterized the AreA nuclear export signal and explored mechanisms that control regulated nuclear export of AreA.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.