Les leucémies aiguës myéloïdes (LAM) représentent un groupe d’hémopathies malignes agressives, de pronostic sombre en dépit des traitements intensifs actuellement proposés. Malgré une grande hétérogénéité clinique et moléculaire, les cellules de LAM sont caractérisées par l’activation de voies de signalisation essentielles à leur prolifération et leur survie, comme par exemple celle du complexe mTORC1 (mammalian target of rapamycin complex 1). Cependant, l’utilisation clinique d’inhibiteurs tels que la rapamycine ou des inhibiteurs catalytiques s’est avérée décevante, ce qui suggère qu’il n’y a pas d’addiction oncogénique à mTORC1 dans les LAM. Au cours de ce travail, nous avons démontré que l’activation de mTORC1 est au contraire une condition nécessaire à l’induction de la mort cellulaire en réponse à l’activation d’AMPK (AMP-activated protein kinase), établissant une relation de létalité synthétique entre ces deux voies. Pour cela, nous avons utilisé un nouveau composé activateur spécifique d’AMPK, le GSK621. En invalidant la sous-unité catalytique AMPKα1 par ARN interférence ou par le système CRISPR/Cas9, nous avons démontré que les effets antileucémiques de ce composé sont bien dépendants de l’activation d’AMPK. Nous avons observé que ce composé favorise l’autophagie, et que ce processus est impliqué dans la mort des cellules leucémiques puisque l’inhibition des protéines ATG5 ou ATG7 a un effet protecteur sur les cellules leucémiques. Les effets antileucémiques du composé GSK621 ont été confirmés sur des cellules primaires, ainsi que sur un panel de vingt lignées de LAM, et dans un modèle murin de xénogreffe. De façon intéressante, l’activation d’AMPK pourrait également compromettre la survie des cellules souches leucémiques, comme en atteste l’atténuation du potentiel clonogénique en méthylcellulose de cellules murines transformées par MLL-ENL ou FLT3-ITD. Nous avons observé que le composé GSK 621 n’avait pas de toxicité envers les progéniteurs hématopoïétiques normaux, ouvrant ainsi une fenêtre thérapeutique intéressante. Comme l’activation d’AMPK conduit dans de nombreux modèles cellulaires à l’inhibition de mTORC1, et comme l’activation de mTORC1 est observée dans les cellules de LAM mais pas dans les progéniteurs hématopoïétiques normaux, nous avons proposé l’hypothèse que le niveau d’activation de mTORC1 déterminait les effets de l’activateur d’AMPK. Pour cela, nous avons inhibé mTORC1 dans les cellules leucémiques d’une part, et activé mTORC1 dans les progéniteurs normaux d’autre part. De façon inattendue, mTORC1 échappe au contrôle d’AMPK dans les LAM, et nous avons observé que l’activation de mTORC1 est une condition nécessaire et suffisante pour que le composé GSK621 entraîne la mort des cellules. Le substrat moléculaire de cette létalité synthétique est le facteur de transcription proapoptotique ATF4, dont la transcription est favorisée par mTORC1, et la traduction par AMPK via la phosphorylation d’eIF2A. Ces travaux proposent donc que malgré l’absence d’addiction oncogénique, l’activation de mTORC1 dans les LAM représente une opportunité thérapeutique originale via une relation de létalité synthétique avec l’activation d’AMPK. Ils constituent un rationnel au développement clinique d’activateurs d’AMPK dans les LAM, voire dans d’autres cancers ayant une activation constitutive de mTORC1. / Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis despite intensive treatments. Virtually all recurrent molecular alterations in AML functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as in clinical trials, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR/Cas9 and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, and blocking autophagy via shRNA-mediated knockdown of ATG5 or ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation between AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1, sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. The same synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. We further observed that the level of ATF4 protein is under a transcriptionnal control by mTORC1 and a translational control by AMPK (through eIF2A), and explains the synthetic lethal relationship between AMPK and mTORC1. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials.

Les leucémies aigues myéloïdes (LAM) sont des hémopathies malignes de mauvais pronostic dont les thérapies actuelles ne permettent d’obtenir des taux de survie à 5 ans chez les adultes que d’environ 40%. Par conséquent, il est nécessaire d’approfondir nos connaissances concernant les mécanismes d’oncogenèse pour développer de nouvelles approches thérapeutiques. Malgré leur hétérogénéité clinique et biologique, les LAM ont certaines caractéristiques communes comme l’activation de la voie de signalisation mTORCl qui est détectée dans la plupart des échantillons de LAM. MTORCl contrôle la survie, la croissance et la prolifération cellulaire, notamment via le contrôle de la traduction des ARNm et donc de la synthèse protéique. Au cours de ce travail, nous montrons qu’il existe, dans les LAM, une dérégulation de mTORCl qui explique les limites des effets anti-leucémiques observés avec la rapamycine (un inhibiteur allostérique de mTORCl) et qui est médiée en partie par l’activité de la sérine thréonine kinase Pim2, qui contrôle la phosphorylation de la cible de mTORCl, la protéine 4E-BP1. Cependant, cibler directement la traduction produit des effets anti-leucémiques importants, ce que nous avons montré en utilisant une molécule inhibant spécifiquement le complexe d’initiation de la traduction, le 4EGI-l. EIF4E est essentiel à l’initiation de la traduction et nous avons montré sa surexpression au niveau protéique dans la plupart des échantillons de LAM au diagnostic par comparaison à des cellules hématopoïétiques normales CD34+. Bien que son niveau d’expression n’ait pas de valeur pronostique intrinsèque, ce résultat suggère un potentiel important au blocage de la traduction dans la plupart des cas de LAM. Dans la perspective d’inhiber mTORCl, nous avons activé la voie LKBl/AMPK par la metformine, ce qui a induit des effets anti-leucémiques in vitro et in vivo via une modification du métabolisme cellulaire avec en particulier une inhibition de la synthèse de protéines oncogéniques. La metformine n’étant pas un candidat en thérapeutique dans les LAM du fait d’un index thérapeutique trop étroit, de nouvelles molécules modulant la voie LKBl/AMPK sont en cours de développement. Enfin, nous avons étudié le rôle de la sérine thréonine kinase Pim2, qui contrôle la traduction protéique et la survie dans les cellules de LAM Flt3-ITD+. Nous avons de plus montré que la sur-expression de Pim2 constitue un nouveau mécanisme de résistance aux inhibiteurs de Flt3 et représente donc une cible thérapeutique prometteuse dans cette catégorie de LAM. L’étude de la voie mTORCl et de la traduction permet donc d’envisager de multiples perspectives thérapeutiques dans les LAM dont certaines sont déjà en cours de développement clinique. / Acute myeloid leukemia (AML) are hematological malignancies with adverse prognosis in which therapies only gives 40% survival within 5 years in adults. Hence, it is important to increase our knowledge regarding oncogenesis to further develop new therapeutic approaches. Despite their clinical and biological heterogeneity, AML have in common the constitutive activation of mTORC1 signaling which is detected in most AML samples. MTORC1 controls cell survival, growth and proliferation, in particular through control of mRNA translation and protein synthesis. During this work, we show, in AML, that mTORC1 is deregulated which explain the poor effects observed with rapamycin (a mTORC1 allosteric inhibitor) and is partially mediated by the serine/threonine kinase Pim-2 which controls the mTORC1 target 4E-BP1. Nevertheless, directly targeting translation, using a specific translation initiation inhibitor named 4EGI-1, have important anti leukemic effects. EIF4E is described as essential in translation initiation and we show its protein overexpression in most AML samples at diagnosis compared with normal hematopoietic CD34+ cells. Whereas eIF4E level expression has no prognostic impact, this result suggests an important potential for treatment targeting translation initiation in AML. In our purpose of inhibiting mTORC1, we were able to activate LKB1/AMPK signaling pathway with metformin, which induces anti leukemic effects in vitro and in vivo through in particular oncogenic protein translation inhibition. Metformin is not a good AML therapeutic candidate because of a narrow therapeutic index, new compound targeting LKB1/AMPK are in development. Finally, we studied the role of the serine/threonine kinase Pim-2 and show that it controls protein translation and FLT3-ITD+ AML cells survival. Furthermore, we show that Pim-2 overexpression is a new mechanism of Flt3 inhibitors resistance and represent a new promising therapeutic target in this AML subtype. Overall, mTORC1 and protein translation study in AML show multiple therapeutics perspective, some of them are already in clinical development.

Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of the plant secondary metabolites, isoflavonoids. These phenolic compounds, which are well-known for their multiple biological effects, are produced mostly in leguminous plants (family Fabaceae). However, at least 225 of them have also been described in 59 other families, without any knowledge of orthologues to hitherto known IFS genes from legumes (with the single exception of sugar beet - Beta vulgaris, from the family Chenopodiaceae). In view of these facts, this masters thesis has focused on two main objectives: (1) to identify isoflavone synthase genes in selected leguminous and non-leguminous plants exploiting the PCR strategy with degenerate and non-degenerate primers, and (2) to find a system for the verification of the correct function of these genes. Our methodology for the identification of IFS orthologues was successfully demonstrated in the case of two examined legumes - Phaseolus vulgaris L. and Pachyrhizus tuberosus (Lam.) Spreng, in the genomic DNA of which the complete IFS sequences have been newly identified. To design a procedure for ascertaining the correct function of these genes and others once they have been completely described, a pilot study with IFS from Pisum sativum L. (CYP93C18; GenBank number...

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<p dir="ltr">Sweetpotato (<i>Ipomoea batatas</i> L.) is a staple crop that provides nutritional benefits to humans globally, but it is subjected to yield loss when competing with weeds, especially during the early stage of establishment. Despite increased organic sweetpotato production in the United States, growers face challenges with limited weed management options and often resort to time-consuming and costly cultivation and hand-weeding. To address this challenge, experiments were developed to determine (1) the effect of sweetpotato cultivar on the critical weed-free period, (2) the effects of in-row plant spacing and cultivar selection on weed suppression and sweetpotato yield, and (3) the impact of buckwheat and silage tarps for row-middle weed control. 1) In 2022, field research was conducted at the Samuel G. Meigs Horticulture Research Farm (Meigs), Lafayette, IN, and at the Southwest Purdue Agricultural Center (SWPAC), Vincennes, IN to estimate the critical weed-free period for ‘Covington’, ‘Murasaki’, and ‘Monaco’ in the Midwest. The experiment was a split-plot design, with weed-free interval treatments as the main plot factor and cultivar as the subplot factor. Weeds were removed by hand and allowed to establish and compete with the crop beginning at 0, 14, 21, 28, 35, or 42 days after transplanting (DAP). As weed-free interval increased from 0 to 42 DAP, predicted total yield increased from 19 kg ha^-1 to 20,540 kg ha^-1for Covington, 3 kg ha^-1 to 11,407 kg ha^-1 for Monaco, and 125 kg ha^-1to 13,460 kg ha^-1for Murasaki at the Lafayette location. A threshold of ≤10% total yield reduction was achieved by maintaining sweetpotatoes weed-free 24 DAP for Covington, 20 DAP for Murasaki, and 33 DAP for Monaco. 2) In 2022 and 2023, studies were conducted at Meigs, Lafayette, IN and SWPAC, Vincennes, IN to evaluate in-row plant spacing and cultivars for weed control and sweetpotato yield. The experiment was a split-split plot design, with in-row spacings of 20, 30, and 40 cm as the main plot factor, weeding frequency (‘critical weed-free period’ and ‘weed free’) as the subplot factor, and sweetpotato cultivar (‘Covington’ and ‘Monaco’) as the sub-subplot factor. However, in 2022, we evaluated only in-row spacing and weeding frequency because of poor establishment of Monaco. In-row spacing had no significant effect on weed densities at 4, 5, and 6 WAP. As in-row spacing increased from 20 to 40 cm, total sweetpotato yield pooled across both locations in 2023 decreased from 30,223 to 21,209 kg ha^-1 for Covington and 24,370 to 20,848 kg ha^-1 for Monaco, however, jumbo yield increased for both cultivars. Findings from this study suggest that an in-row spacing of 20 cm may provide greater yield than the standard spacing, 30 cm, for both Monaco and Covington cultivars and could reduce weed interference through more rapid sweetpotato canopy closure. 3) The experiment was a randomized complete block design, with three row-middle treatments [tarp, buckwheat, and cultivation] and four replicates. Row-middle treatments were established immediately after transplanting ‘Covington’ slips 30 cm apart into raised bed plots consisting of a single row 6 m long and 2 m apart on-center. Buckwheat was planted three weeks after transplanting (WAP) at a rate of 108 kg ha^-1 in row-middle. Row-middles for the tarp treatment were covered for the entire growing season. Weed density at 6 WAP was 184 plants m^-2 for the buckwheat, and 162 plants m^-2 for the cultivation treatments. Total yield was 11,050 kg ha^-1 for the buckwheat, 19,790 kg ha^-1 for the cultivation, and 17,810 kg ha^-1 for the tarping treatments. Tarping effectively suppressed weeds and produced sweetpotato yields comparable to cultivation indicating potential for organic growers. Buckwheat yields were lower than those from tarping and cultivation.</p>

Increasing awareness of sustainable building materials has led to interest in enhancing the structural performance of engineered wood products. Wood is a sustainable, renewable material, and the increasing use of wood in construction contributes to its sustainability. Multi-layer wooden panels are one type of engineered wood product used in construction. There are various techniques to assemble multi-layer wooden panels into prefabricated, load-bearing construction elements. Assembly techniques considered in the earliest stages of this research work were laminating, nailing, stapling, screwing, stress laminating, doweling, dovetailing, and wood welding. Cross-laminated timber (CLT) was found to offer some advantages over these other techniques. It is cost-effective, not patented, offers freedom of choice regarding the visibility of surfaces, provides the possibility of using different timber quality in the same panel at different points of its thickness, and is the most well-established assembly technique currently used in the industrial market. Building upon that foundational work, the operational capabilities of CLT were further evaluated by creating panels with different layer orientations. The mechanical properties of CLT panels constructed with layers angled in an alternative configuration produced on a modified industrial CLT production line were evaluated. Timber lamellae were adhesively bonded in a single-step press procedure to form CLT panels. Transverse layers were laid at a 45° angle instead of the conventional 90° angle with respect to the longitudinal layers’ 0° angle. Tests were carried out on 40 five-layered CLT panels, each with either a ±45° or a 90° configuration. Half of these panels were evaluated under bending: out-of-plane loading was applied in the principal orientation of the panels via four-point bending. The other twenty were evaluated under compression: an in-plane uniaxial compressive loading was applied in the principal orientation of the panels. Quasi-static loading conditions were used for both in- and out-of-plane testing to determine the extent to which the load-bearing capacity of such panels could be enhanced under the current load case. Modified CLT showed higher stiffness, strength, and fifth-percentile characteristics, values that indicate the load-bearing capacity of these panels as a construction material. Failure modes under in- and out-of-plane loading for each panel type were also assessed. Data from out-of-plane loading were further analysed. A non-contact full-field measurement and analysis technique based on digital image correlation (DIC) was utilised for analysis at global and local scales. DIC evaluation of 100 CLT layers showed that a considerable part of the stiffness of conventional CLT is reduced by the shear resistance of its transverse layers. The presence of heterogeneous features, such as knots, has the desirable effect of reducing the propagation of shear fraction along the layers. These results call into question the current grading criteria in the CLT standard. It is suggested that the lower timber grading limit be adjusted for increased value-yield. The overall experimental results suggest the use of CLT panels with a ±45°-layered configuration for construction. They also motivate the use of alternatively angled layered panels for more construction design freedom, especially in areas that demand shear resistance. In addition, the design possibility that such 45°-configured CLT can carry a given load while using less material than conventional CLT suggests the potential to use such panels in a wider range of structural applications. The results of test production revealed that 45°-configured CLT can be industrially produced without using more material than is required for construction of conventional 90°-configured panels. Based on these results, CLT should be further explored as a suitable product for use in more wooden-panel construction. / <p>External cooperation: Martinson Group AB and Research Institutes of Sweden (RISE)</p>

Thesis(M.S.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.