Academic literature on the topic 'Glycerol signaling'

SUMMARY The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects.

Background: Colon cancer begins in the large intestine (colon) and is aggressive due to late diagnosis, so there is a poor prognosis and higher mortality rates, as reported. Colon cancer has become a vital research area requiring more investigation of cellular signaling in its initiation and development. Aim: The study aimed to investigate the biological effects of Glycerol in cell proliferation and the possible regulation of cellular signaling by exogenous treatment of Glycerol in colon cancer cells compared with colon mucosal epithelial cells. Materials and Methods: The influence of Glycerol on cell viability rate was monitored by inverted microscopy, and the number of living cells was assessed upon incubation with different concentrations of Glycerol. We further inspected the apoptotic rate of CaCo-2 cells by using Annexin-V staining by flow cytometry. Moreover, we achieved the expression profile of lysosomal-associated proteins, LAMP-1 and LAMP-2, in treated cells using qRT-PCR and flow cytometry. Finally, we monitored the released pro-inflammatory cytokines and anti-inflammatory in response to Glycerol treatment using ELISA assay. Results: Our results showed that Glycerol treatment could prevent cancer cell proliferation without any detectable cytotoxic occurrence in the normal cells. Interestingly, we evidenced that Glycerol targets and breaks down the lysosomal activities by inhibiting the expression profile of both LAMP-1 and LAMP-2. Furthermore, Glycerol treatment successfully adjusted the production of IL-6 and IL-8 as pro-inflammatory cytokines while stimulating the production of anti-inflammatory cytokines, IL-4 and IL-10, in a time-dependent manner. Conclusion: These data provide evidence for the anti-cancer properties of Glycerol in colon cancer cells via targeting lysosomal activities and disturbance of the degradation events in colon cancer cells.

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as “catabolite repression,” allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named “metabolic contest” for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This “metabolic contest” depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.

ABSTRACT Yeast cells adapt to hyperosmotic shock by accumulating glycerol and altering expression of hundreds of genes. This transcriptional response of Saccharomyces cerevisiae to osmotic shock encompasses genes whose products are implicated in protection from oxidative damage. We addressed the question of whether osmotic shock caused oxidative stress. Osmotic shock did not result in the generation of detectable levels of reactive oxygen species (ROS). To preclude any generation of ROS, osmotic shock treatments were performed in anaerobic cultures. Global gene expression response profiles were compared by employing a novel two-dimensional cluster analysis. The transcriptional profiles following osmotic shock under anaerobic and aerobic conditions were qualitatively very similar. In particular, it appeared that expression of the oxidative stress genes was stimulated upon osmotic shock even if there was no apparent need for their function. Interestingly, cells adapted to osmotic shock much more rapidly under anaerobiosis, and the signaling as well as the transcriptional response was clearly attenuated under these conditions. This more rapid adaptation is due to an enhanced glycerol production capacity in anaerobic cells, which is caused by the need for glycerol production in redox balancing. Artificially enhanced glycerol production led to an attenuated response even under aerobic conditions. These observations demonstrate the crucial role of glycerol accumulation and turgor recovery in determining the period of osmotic shock-induced signaling and the profile of cellular adaptation to osmotic shock.

During development, nephron progenitor cells (NPC) are induced to differentiate by WNT9b signals from the ureteric bud. Although nephrogenesis ends in the perinatal period, acute kidney injury (AKI) elicits repopulation of damaged nephrons. Interestingly, embryonic NPC infused into adult mice with AKI are incorporated into regenerating tubules. Since WNT/β-catenin signaling is crucial for primary nephrogenesis, we reasoned that it might also be needed for the endogenous repair mechanism and for integration of exogenous NPC. When we examined glycerol-induced AKI in adult mice bearing aβ-catenin/TCF reporter transgene, endogenous tubular cells reexpressed the NPC marker, CD24, and showed widespreadβ-catenin/TCF signaling. We isolated CD24+cells from E15 kidneys of mice with the canonical WNT signaling reporter. 40% of cells responded to WNT3ain vitroand when infused into glycerol-injured adult, the cells exhibitedβ-catenin/TCF reporter activity when integrated into damaged tubules. When embryonic CD24+cells were treated with aβ-catenin/TCF pathway inhibitor (IWR-1) prior to infusion into glycerol-injured mice, tubular integration of cells was sharply reduced. Thus, the endogenous canonicalβ-catenin/TCF pathway is reactivated during recovery from AKI and is required for integration of exogenous embryonic renal progenitor cells into damaged tubules. These events appear to recapitulate the WNT-dependent inductive process which drives primary nephrogenesis.

Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the Toll-like receptor-4 (TLR4) receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-κB and its downstream gene monocyte chemoattractant protein-1(MCP-1); in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor Nrf2 and its downstream gene heme oxygenase-1 (HO-1). TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histological injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4+/+ and TLR4−/− mice. We conclude that 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; and 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in virtually all other AKI models studied to date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what occurs in vivo.

Glucose metabolism promotes insulin secretion in β-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes β-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the β-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in β-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in β-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.

Hydrogen sulfide (H2S) and inorganic polysulfides are important signaling molecules; however, little is known about their role in the adipose tissue. We examined the effect of H2S and polysulfides on adipose tissue lipolysis. H2S and polysulfide production by mesenteric adipose tissue explants in rats was measured. The effect of Na2S and Na2S4, the H2S and polysulfide donors, respectively, on lipolysis markers, plasma non-esterified fatty acids (NEFA) and glycerol, was examined. Na2S but not Na2S4 increased plasma NEFA and glycerol in a time- and dose-dependent manner. Na2S increased cyclic AMP but not cyclic GMP concentration in the adipose tissue. The effect of Na2S on NEFA and glycerol was abolished by the specific inhibitor of protein kinase A, KT5720. The effect of Na2S on lipolysis was not abolished by propranolol, suggesting no involvement of β-adrenergic receptors. In addition, Na2S had no effect on phosphodiesterase activity in the adipose tissue. Obesity induced by feeding rats a highly palatable diet for 1 month was associated with increased plasma NEFA and glycerol concentrations, as well as greater H2S production in the adipose tissue. In conclusion, H2S stimulates lipolysis and may contribute to the enhanced lipolysis associated with obesity.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride–glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

The T cell receptor (TCR) activation induced signaling cascade is a major driver of T cell effector responses such as cytokine production and actin cytoskeletal rearrangement. Characterizing chemical modulators of this pathway has the benefits of both revealing basic science knowledge about these signaling processes and providing foundation for development of novel therapeutics. Glycerol Monolaurate (GML) is a naturally occurring fatty acid monoester that is found as a monoglyceride in human breast milk and coconut oil. It is widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a wide range of bacteria, fungi, and enveloped viruses. Because of this, GML has been developed as a preventative for menstrual associated Toxic Shock Syndrome, and is being tested to prevent HIV transmission and superficial skin infections. Interestingly, GML suppresses mitogen induced lymphocyte proliferation and inositol triphosphate production, suggesting that GML has immunomodulatory functions. This thesis mechanistically examined how GML affects human primary T cells. Chapter III describes how GML potently altered order and disorder dynamics in the plasma membrane that resulted in reduced membrane-localized clustering of the proteins LAT, PLC-γ, and AKT, events integral for proper TCR signal propagation. Altered membrane signaling events induced selective inhibition of TCR-induced signaling events. Specifically GML reduced the phosphorylation of the regulatory P85 subunit of PI3K, and AKT and abrogated calcium influx. Functionally, GML treatment potently reduced TCR-induced production of the cytokines IL-2, IFN-γ, TNF-α, and IL-10. Chapter V shows that GML causes the mis-localization of the ARPC3 subunit of the Arp2/3 complex that leads to the formation of abnormal filopodia structures, and reduced cellular adhesion. Chapter V shows that human serum albumin binds directly to GML on the 12 carbon acyl chain. This interaction reverses GML induced suppression of TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane, AKT phosphorylation, and cytokine production. These findings establish GML as a T cell suppressive agent in addition to an antimicrobial agent. This observation reveals the potential role of naturally occurring GML in human breast milk in the formation of microbiota and immune tolerance in the infant gastrointestinal tract. It also allows for optimization of the current applications of GML in various commercial products and therapeutic strategies. Finally this information provides the rationale to investigate GML in new remedial avenues as a topical agent to treat excessive inflammation in the skin, and vaginal and gut mucosal regions.

Plants use several strategies to defend themselves against microbial pathogens. These include basal resistance, which is induced in response to pathogen encoded effector proteins, and resistance (R) protein-mediated resistance that is activated upon direct or indirect recognition of pathogen encoded avirulence protein(s). The activation of Rmediated signaling is often associated with generation of a signal, which, upon its translocation to the distal uninfected parts, confers broad-spectrum immunity against related or unrelated pathogens. This phenomenon known as systemic acquired resistance (SAR) is one of the well-established forms of induced defense response. However, the molecular mechanism underlying SAR remains largely unknown. Induction of plant defense is often associated with a fitness cost, likely because it involves reprogramming of the energy-providing metabolic pathways. Glycerol metabolism is one such pathway that feeds into primary metabolism, including lipid biosynthesis. In this study, I evaluated the role of glycerol-3-phosphate (G3P) in host-pathogen interaction. Inoculation with the hemibiotrophic fungal pathogen Colletotrichum higginsianum led to increased accumulation of G3P in wild-type plants. Mutants impaired in biosynthesis of G3P showed enhanced susceptibility, suggesting a correlation between G3P levels and basal defense. Conversely, increased biosynthesis of G3P correlated with enhanced resistance. The Arabidopsis genome encodes one copy of glycerol kinase (GK), which catalyzes phosphorylation of glycerol to G3P, and five copies of G3P dehydrogenase (G3Pdh), which catalyze reduction of dihydroxyacetone phosphate to G3P. Analysis of plants mutated in various G3Pdh's showed that plastidal lipid biosynthesis was only dependent on the GLY1 isoform but the pathogen induced G3P pool required the function of GLY1 and two other G3Pdh isoforms. Interestingly, compromised G3P biosynthesis in GK and G3Pdh mutants also compromised SAR, which was restored when G3P was provided exogenously. Detailed biochemical analysis showed that G3P was transported to distal tissues and that this process was dependent on a lipid transfer protein, DIR1. Together, these results show that G3P plays an important role in both basal- and induced-defense responses.

Oleic acid (18:1), a monounsaturated fatty acid (FA), is synthesized upon desaturation of stearic acid (18:0) and this reaction is catalyzed by the plastidal enzyme stearoyl-acyl carrier protein-desaturase (SACPD). A mutation in the SSI2/FAB2 encoded SACPD lowers 18:1 levels, which correlates with induction of various resistance (R) genes and increased resistance to pathogens. Genetic and molecular studies have identified several suppressors of ssi2 which restore altered defense signaling either by normalizing 18:1 levels or by affecting function(s) of a downstream component. Characterization of one such ssi2 suppressor mutant showed that it is required downstream of low 18:1-mediated constitutive signaling and partially restores altered defense signaling in the ssi2 mutant. Molecular and genetic studies showed that the second site mutation was in the Nitric Oxide Associated (NOA) 1 gene, which is thought to participate in NO biosynthesis. Consistent with this result, ssi2 plants accumulated high levels of NO and showed an altered transcriptional profile of NO-responsive genes. Interestingly, the partial defense phenotypes observed in ssi2 noa1 plants were completely restored by an additional mutation in either of the two nitrate reductases NIA1 or NIA2. This suggested that NOA1 and NIA proteins participated in NO biosynthesis in an additive manner. Biochemical studies showed that 18:1 physically bound NOA1, in turn leading to its degradation in a protease-dependent manner. In concurrence, overexpression of NOA1 did not promote NO-derived defense signaling in wild-type plants unless 18:1 levels were lowered. Subcellular localization showed that NOA1 and the 18:1-synthesizing SSI2 were present in close proximity within the nucleoids of chloroplasts. Indeed, pathogen- or low 18:1- induced accumulation of NO was primarily detected in the chloroplasts and their nucleoids. Together, these data suggested that 18:1 levels regulate NO synthesis and thereby NO-mediated retrograde signaling between the nucleoids and the nucleus. Since cellular pools of glycerol-3-phosphate (G3P) regulate 18:1 levels, I next analyzed the relationship between G3P and 18:1. Interestingly, unlike 18:1, an increased G3P pool was associated with enhanced systemic immunity in Arabidopsis. This was consistent with G3P-mediated transcriptional reprogramming in the distal tissues. To determine mechanism(s) underlying G3P-conferred systemic immunity, I analyzed the interaction between G3P and a lipid transfer protein (LTP), DIR1. In addition, I monitored localization of DIR1 in both Arabidopsis as well as tobacco. Contrary to its predicted apoplastic localization, DIR1 localized to endoplasmic reticulum and plasmodesmata. The symplastic localization of DIR1 was confirmed using several different assays, including co-localization with plasmodesmatal-localizing protein, plasmolysis and protoplast-based assays. Translocation assays showed that G3P increased DIR1 levels and translocated DIR1 to distal tissues. Together, these results showed that G3P and DIR1 are present in the symplast and their coordinated transport into distal tissues is likely essential for systemic immunity. In conclusion, this work showed that low 18:1-mediated signaling is mediated via NO, synthesis of which is likely initiated in the plastidal nucleoids. In addition, my work shows that G3P functions as an independent signal during systemic signaling by mediating translocation of the lipid transfer protein, DIR1.

Oleic acid (18:1) is one of the important monounsaturated fatty acids, which is synthesized upon desaturation of stearic acid and this reaction is catalyzed by the SSI2 encoded stearoyl-acyl-carrier-protein-desaturase. A mutation in SSI2 leads to constitutive activation of salicylic acid (SA)-mediated defense responses. Consequently, these plants accumulate high levels of SA and show enhanced resistance to bacterial and oomycete pathogens. Replenishing 18:1 levels in ssi2 plants, via a second site mutation in GLY1 encoded glycerol-3-phosphate (G3P) dehydrogenase, suppresses all the ssi2-triggered phenotypes. Study of mechanism(s) underlying gly1-mediated suppression of ssi2 phenotypes showed that 18:1 levels are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of defense signaling pathways. To establish a role for 18:1 and G3P during host defense, interaction between Colletotrichum higginsianum and Arabidopsis was studied. Resistance to C. higginsianum correlated with host G3P levels. The gly1 plants showed increased susceptibility while act1 plants, defective in utilization of G3P, showed enhanced resistance. Plant overexpessing GLY1 showed enhanced resistance in both wild type as well as camalexin deficient backgrounds. Together, these results suggested that G3P conferred resistance acted downstream or independent of camalexin. Exogenous application of glycerol lowered 18:1 levels and produced ssi2-like phenotypes in wild-type plants. Furthermore, glycerol application or the ssi2 mutation produced similar phenotypes in fatty acid desaturation mutants and mutants defective in SA/resistance gene signaling. Expression studies showed that ssi2 phenotypes were likely due to increased expression of resistance genes. Epistatic analysis suggested that certain components of SA pathway had redundant function and were required for 18:1-regulated signaling.

xii, 41 p. : ill.
Cells convey information through signaling pathways. Distinct signaling pathways often rely on similar mechanisms and may even use the same molecules. With a variety of signals conveyed by pathways that share components, how does the cell maintain the integrity of each pathway? Budding yeast provides an example of multiple signaling pathways utilizing the same components to transduce different signals. The mating pathway, the high osmolarity glycerol (HOG) pathway and the filamentous growth (FG) pathway each respond to different environmental conditions and generate unique cellular responses. Despite the individuality of the pathways, they each contain a core group of the same signaling proteins. How does the cell generate a variety or responses utilizing the same group of proteins? Both the mating and HOG pathways utilize scaffolding factors that concentrate pathway components to the location of activation and in the case of the mating pathway alter the kinetics of the interaction. In addition, negative regulatory mechanisms operate in both the mating and HOG pathways. These negative regulatory mechanisms are understood in detail for the mating pathway but not for the HOG pathway. Mechanisms for providing specificity for the FG pathway are as yet unknown. The purpose of this work is to elucidate the mechanisms that provide specificity to the FG pathway. The search for specificity factors was done through both a random mutagenesis screen and a synthetic genetic array screen, looking for mutants in which activation of the FG pathway led to inappropriate activation of the HOG pathway. The random mutagenesis screen resulted in a large number of mutants that I organized into five complementation groups. The identity of the gene mutated in the largest complementation group was sought using a variety of methods including complementation with the yeast deletion collection and whole genome sequencing. A synthetic genetic array was screened as an alternative method to identify genes necessary for FG pathway specificity. These experiments have resulted in a list of candidate genes, but thus far have not yet led to any discernable mechanism for maintenance of FG pathway specificity.
Committee in charge: Karen Guillemin, Chairperson; George F. Sprague Jr., Advisor; Tom Stevens, Member; Tory Herman, Member; Diane Hawley, Outside Member

Systemic acquired resistance (SAR) is a form of inducible defense response triggered upon localized infection that confers broad-spectrum disease resistance against secondary infections. Several factors are known to regulate SAR and these include phenolic phytohormone salicylic acid (SA), phosphorylated sugar glycerol-3-phosphate (G3P), and dicarboxylic acid azelaic acid (AzA). This study evaluated a role for free radicals nitric oxide (NO) and reactive oxygen species (ROS) in SAR. Normal accumulation of both NO and ROS was required for normal SAR and mutations preventing NO/ROS accumulation and/or biosynthesis compromised SAR. A role for NO and ROS was further established using pharmacological approaches. Notably, both NO and ROS conferred SAR in a concentration dependent manner. This was further established using genetic mutants that accumulated high levels of NO. NO/ROS acted upstream of G3P and in parallel to SA. Collectively, these results suggest that NO and ROS are essential components of the SAR pathway.

Trypanosoma brucei, un parasite extracellulaire responsable de la trypanosomiase africaine, doit s’adapter à différents environnements dans ses hôtes mammifères et l’insecte vecteur (la mouche tsétsé). Dans le sang des mammifères, le glucose est la principale source de carbone soutenant une croissance rapide du parasite, en alimentant son métabolisme et la production d'ATP. Lorsque les formes sanguines prolifératives « slender » atteignent des densités cellulaires élevées, un quorum sensing induit leur différenciation en formes non réplicatives « stumpy » (stumpy-QS). Ces formes stumpy-QS protègent l’hôte d’une parasitémie létale et sont également compétentes pour la transmission à la mouche tsétsé. Cependant, le glucose peut être remplacé par le glycérol pour alimenter le métabolisme central du parasite, suggérant un rôle significatif in vivo. Cela s'aligne avec la démonstration récente que les trypanosomes résident principalement dans les espaces extravasculaires de la peau et du tissu adipeux, où le glycérol interstitiel est 5 à 20 fois plus concentré que dans le plasma. Le glycérol est produit par les adipocytes via la glycolyse et la lipolyse, cette dernière induite par les trypanosomes, pourrait protéger l'hôte contre l'infection. Ces données suggèrent que les interactions entre les adipocytes et les trypanosomes, potentiellement médiées par le glycérol, jouent un rôle dans le cycle de vie du parasite.Cette thèse explore l’impact du glycérol sur les formes sanguines slender de Trypanosoma brucei. Nos résultats ont démontré que le glycérol induit la différenciation des formes prolifératives en formes non réplicatives (stumpy-Glyc) ressemblant aux formes stumpy-QS, mais avec une survie allongée. Des conditions similaires à celles des tissus (4 mM glucose, 0,2-0,5 mM glycérol) induisent la production de formes intermédiaires prolifératives (intermédiaire-Glyc) capables de se différencier in vitro en formes procycliques (parasites de l’intestin de l'insecte vecteur) et d’infecter les mouches tsétsé. De plus, le glycérol prolonge considérablement la durée de vie des formes stumpy-QS induites par le quorum sensing. Ces données nous ont conduit à proposer un modèle révisé de la transmission du parasite de l’hôte mammifère à l’insecte vecteur, où les formes stumpy-QS protègent l'hôte contre une parasitémie élevée, tandis que les formes intermédiaires-Glyc prolifératives et les formes stumpy à longue durée de vie, induites par le glycérol des adipocytes, assurent la transmission à l’insecte vecteur.Un autre aspect de ma thèse concerne la dissection de la voie de signalisation impliquée dans la différenciation induite par le glycérol. En exploitant la durée de vie prolongée des cellules stumpy-Glyc en culture, nous avons sélectionné des mutants résistants à la différenciation induite par le glycérol. L'analyse génomique comparative entre ces mutants a permis d'identifier des mutations candidates associées au phénotype de résistance. Une mutation a notamment été trouvée dans le gène de la sous-unité régulatrice de la protéine kinase A (PKAR), dont le rôle dans la voie de signalisation a été ensuite validé.Enfin, nous avons exploré la capacité de T. brucei à métaboliser le glycérol sécrété par les adipocytes, même en présence d'un excès de glucose. Pour ce faire, nous avons utilisé un système de co-culture in vitro permettant d'analyser les interactions entre les trypanosomes parentaux ou mutants et les adipocytes. Le profilage métabolique par résonance magnétique nucléaire (RMN) couplé à des approches de marquage au 13C a permis de tracer les métabolites produits par les adipocytes et les trypanosomes. Nos données ont montré que T. brucei utilise efficacement le glycérol sécrété par les adipocytes pour alimenter son métabolisme central, même en présence de grandes quantités de glucose.En conclusion, ces données ont démontré que le glycérol est un acteur clé dans la biologie de Trypanosoma brucei
Trypanosoma brucei, an extracellular parasite responsible for African trypanosomiasis, must adapt to distinct environments in its mammalian hosts and the tsetse fly vector. In the mammalian bloodstream, glucose serves as the primary carbon source, fueling the parasite's central carbon metabolism and ATP production, which supports its rapid growth. Once the parasites reach high cell densities, a quorum-sensing mechanism induces a transition from proliferative slender forms to growth-arrested stumpy forms (stumpy-QS). These stumpy forms help prevent host mortality by limiting parasitaemia and are primed for transmission to the tsetse fly. However, it has been demonstrated that glycerol can effectively replace glucose in feeding the parasite’s central carbon metabolism, suggesting a significant role in vivo. This aligns with findings that trypanosomes predominantly reside in the extravascular spaces of tissues such as the skin and adipose tissue, where interstitial glycerol concentrations are 5 to 20 times higher than in plasma. Glycerol is released from adipocytes through both lipolysis and lipolysis-independent processes such as glycolysis, and it has been suggested that trypanosome-induced adipocyte lipolysis may even protect the host against trypanosome infection. Together, these data suggest that interactions between adipocytes and trypanosomes, potentially mediated by glycerol, play a critical role in the parasite’s life cycle.This thesis explores the impact of glycerol on bloodstream form (BSF) Trypanosoma brucei. Our findings demonstrated that glycerol induces the differentiation of slender BSF into growth-arrested forms that resemble stumpy-QS, but with enhanced survival. Furthermore, under tissue-like conditions, characterized by glycerol levels between 0.2-0.5 mM and glucose at 4 mM, proliferative intermediate forms were generated, which were capable of differentiating into the insect vector stage (procyclics) and sustaining infections in tsetse flies. Additionally, glycerol extended the lifespan of quorum-sensing-induced stumpy forms, which normally have a limited lifespan of a few days. All these data led us to propose a revised model for transmission, in which quorum sensing-induced stumpy-QS forms protect the host from high parasitaemia, while glycerol from adipocytes induces intermediate-Glyc or long-lived stumpy forms that facilitate transmission to the fly.Another key aspect of my thesis concerns the dissection of the signalling pathway involved in glycerol-induced differentiation. By exploiting the extended lifespan of stumpy-Glyc cells in culture, we selected mutants resistant to glycerol-induced differentiation through extended in vitro culturing in a glycerol-containing medium. Comparative genomic analyses between these mutants and cells grown in glucose, which are sensitive to glycerol-induced differentiation, identified candidate mutations associated with the resistance phenotype. Notably, these mutations were found to affect the protein kinase A regulatory subunit (PKAR), whose role in the signalling pathway was validated.Finally, we explored whether T. brucei can metabolize glycerol secreted by adipocytes even in the presence of excess glucose. To investigate this, we used an in vitro co-culture system using a transwell assay, which allowed us to analyse the interactions between parental and mutant trypanosomes and adipocytes. We examined growth and exometabolome profiles using nuclear magnetic resonance (NMR)-based metabolite profiling, coupled with 13C-labeling to trace specific metabolites. Our data showed that T. brucei efficiently utilized glycerol secreted by adipocytes to support its central carbon metabolism, even when glucose was abundant.Together, these data demonstrated that glycerol is a key player in the biology of Trypanosoma brucei

Abstract Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a glycerol-phospholipid found predominantly in the inner leaflet of eukaryotic plasma membranes (Fig. 1). Although it constitutes less than 0.05% of total cellular phospholipids, PtdIns(4,5)P2 plays a critical role in intracellular signalling. PtdIns(4,5)P2 is best known for its ability to serve as a precursor for the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG). Ins(1,4,5)P3 and DAG are generated from the hydrolysis of PtdIns(4,5)P2 by phospholipases C (PLCs) following agonist stimulation (1, 2). Production of Ins(1,4,5)P3 triggers a transient rise in intracellular calcium (Ca2+), while the generation of DAG activates protein kinase C (PKC) family members.

Introduction Previous research had suggested that benzoxazinoids, a class of defensive metabolites found in maize, wheat, rye, and wild barley, are not only direct insect deterrents, but also influence other areas of plant metabolism. In particular, the benzoxazinoid 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxa- zin-3(4H)- one (DIMBOA) was implicated in: (i) altering plant growth by interfering with auxin signaling, and (ii) leading to the induction of gene expression changes and secondary plant defense responses. The overall goal of this proposal was to identify mechanisms by which benzoxazinoids influence other aspects of plant growth and defense. Specifically, the following hypotheses were proposed to be tested as part of an approved BARD proposal: Benzoxazinoid breakdown products directly interfere with auxin perception Global changes in maize and barley gene expression are induced by benzoxazinoid activation. There is natural variation in the maize photomorphogenic response to benzoxazinoids. Although the initial proposal included experiments with both maize and barley, there were some technical difficulties with the proposed transgenic barley experiments and most of the experimental results were generated with maize. Summary of major findings Previous research by other labs, involving both maize and other plant species, had suggested that DIMBOA alters plant growth by interfering with auxin signaling. However, experiments conducted in both the Chamovitz and the Jander labs using Arabidopsis and maize, respectively, were unable to confirm previously published reports of exogenously added DIMBOA effects on auxin signaling. Nevertheless, analysis of bx1 and bx2 maize mutant lines, which have almost no detectable benzoxazinoids, showed altered responses to blue light signaling. Transcriptomic analysis of maize mutant lines, variation in inbred lines, and responses to exogenously added DIMBOA showed alteration in the transcription of a blue light receptor, which is required for plant growth responses. This finding provides a novel mechanistic explanation of the trade-off between growth and defense that is often observed in plants. Experiments by the Jander lab and others had demonstrated that DIMBOA not only has direct toxicity against insect pests and microbial pathogens, but also induces the formation of callose in both maize and wheat. In the current project, non-targeted metabolomic assays of wildtype maize and mutants with defects in benzoxazinoid biosynthesis were used to identify unrelated metabolites that are regulated in a benzoxazinoid-dependent manner. Further investigation identified a subset of these DIMBOA-responsive compounds as catechol, as well as its glycosylated and acetylated derivatives. Analysis of co-expression data identified indole-3-glycerol phosphate synthase (IGPS) as a possible regulator of benzoxazinoid biosynthesis in maize. In the current project, enzymatic activity of three predicted maize IGPS genes was confirmed by heterologous expression. Transposon knockout mutations confirmed the function of the maize genes in benzoxazinoid biosynthesis. Sub-cellular localization studies showed that the three maize IGPS proteins are co-localized in the plastids, together with BX1 and BX2, two previously known enzymes of the benzoxazinoid biosynthesis pathway. Implications Benzoxazinoids are among the most abundant and effective defensive metabolites in maize, wheat, and rye. Although there is considerable with-in species variation in benzoxazinoid content, very little is known about the regulation of this variation and the specific effects on plant growth and defense. The results of this research provide further insight into the complex functions of maize benzoxazinoids, which are not only toxic to pests and pathogens, but also regulate plant growth and other defense responses. Knowledge gained through the current project will make it possible to engineer benzoxazinoid biosynthesis in a more targeted manner to produce pest-tolerant crops without negative effects on growth and yield.