Production of polyhydroxyalkanoates from oil-containing substrates

Different oil-containing substrates, namely, used cooking oil (UCO), fatty acids-byproduct from biodiesel production (FAB) and olive oil deodorizer distillate (OODD) were tested as inexpensive carbon sources for the production of polyhydroxyalkanoates (PHA) using twelve bacterial strains, in batch experiments. The OODD and FAB were exploited for the first time as alternative substrates for PHA production. Among the tested bacterial strains, Cupriavidus necator and Pseudomonas resinovorans exhibited the most promising results, producing poly-3-hydroxybutyrate, P(3HB), form UCO and OODD and mcl-PHA mainly composed of 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) monomers from OODD, respectively. Afterwards, these bacterial strains were cultivated in bioreactor. C. necator were cultivated in bioreactor using UCO as carbon source. Different feeding strategies were tested for the bioreactor cultivation of C. necator, namely, batch, exponential feeding and DO-stat mode. The highest overall PHA productivity (12.6±0.78 g L-1 day-1) was obtained using DO-stat mode. Apparently, the different feeding regimes had no impact on polymer thermal properties. However, differences in polymer‟s molecular mass distribution were observed. C. necator was also tested in batch and fed-batch modes using a different type of oil-containing substrate, extracted from spent coffee grounds (SCG) by super critical carbon dioxide (sc-CO2). Under fed-batch mode (DO-stat), the overall PHA productivity were 4.7 g L-1 day-1 with a storage yield of 0.77 g g-1. Results showed that SCG can be a bioresource for production of PHA with interesting properties. Furthermore, P. resinovorans was cultivated using OODD as substrate in bioreactor under fed-batch mode (pulse feeding regime). The polymer was highly amorphous, as shown by its low crystallinity of 6±0.2%, with low melting and glass transition temperatures of 36±1.2 and -16±0.8 ºC, respectively. Due to its sticky behavior at room temperature, adhesiveness and mechanical properties were also studied. Its shear bond strength for wood (67±9.4 kPa) and glass (65±7.3 kPa) suggests it may be used for the development of biobased glues. Bioreactor operation and monitoring with oil-containing substrates is very challenging, since this substrate is water immiscible. Thus, near-infrared spectroscopy (NIR) was implemented for online monitoring of the C. necator cultivation with UCO, using a transflectance probe. Partial least squares (PLS) regression was applied to relate NIR spectra with biomass, UCO and PHA concentrations in the broth. The NIR predictions were compared with values obtained by offline reference methods. Prediction errors to these parameters were 1.18 g L-1, 2.37 g L-1 and 1.58 g L-1 for biomass, UCO and PHA, respectively, which indicates the suitability of the NIR spectroscopy method for online monitoring and as a method to assist bioreactor control. UCO and OODD are low cost substrates with potential to be used in PHA batch and fed-batch production. The use of NIR in this bioprocess also opened an opportunity for optimization and control of PHA production process.

Polyhydroxyalkanoates production by photosynthetic mixed cultures

Polyhydroxyalkanoates (PHAs) are natural biologically synthesized polymers that have been the subject of much interest in the last decades due to their biodegradability. Thus far, its microbial production is associated with high operational costs, which increases PHA prices and limits its marketability. To address this situation, this thesis’ work proposes the utilization of photosynthetic mixed cultures (PMC) as a new PHA production system that may lead to a reduction in operational costs. In fact, the operational strategies developed in this work led to the selection of PHA accumulating PMCs that, unlike the traditional mixed microbial cultures, do not require aeration, thus permitting savings in this significant operational cost. In particular, the first PHA accumulating PMC tested in this work was selected under non-aerated illuminated conditions in a feast and famine regime, being obtained a consortium of bacteria and algae, where photosynthetic bacteria accumulated PHA during the feast phase and consumed it for growth during the famine phase, using the oxygen produced by algae. In this symbiotic system, a maximum PHA content of 20% cell dry weight (cdw) was reached, proving for the first time, the capacity of a PMC to accumulate PHA. During adaptation to dark/light alternating conditions, the culture decreased its algae content but maintained its viability, achieving a PHA content of 30% cdw. Also, the PMC was found to be able to utilize different volatile fatty acids for PHA production, accumulating up to 20% cdw of a PHA co-polymer composed of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (HV) monomers. Finally, a new selective approach for the enrichment of PMCs in PHA accumulating bacteria was tested. Instead of imposing a feast and famine regime, a permanent feast regime was used, thus selecting a PMC that was capable of simultaneously growing and accumulating PHA, being attained a maximum PHA content of 60% cdw, the highest value reported for a PMC thus far. The results presented in this thesis prospect the utilization of cheap, VFA-rich fermented wastes as substrates for PHA production, which combined with this new photosynthetic technology opens up the possibility for direct sunlight illumination, leading to a more cost-effective and environmentally sustainable PHA production process.

Chlorite Dismutase from perchlorate reducing bacteria. A potential biocatalyst to eliminate chlorinated species from polluted water resources and industrial wastes

Magnetospirillum (M.) sp. strain Lusitani, a perchlorate reducing bacteria (PRB), was previously isolated from a wastewater treatment plant and phylogenetic analysis was performed to classify the isolate. The DNA sequence of the genes responsible for perchlorate reduction and chlorite dismutation was determined and a model was designed based on the physiological roles of the proteins involved in the pcr-cld regulon. Chlorite dismutase (Cld) was purified from Magnetospirillum sp. strain Lusitani cells grown in anaerobiosis in the presence of perchlorate. The protein was purified up to electrophoretic grade using HPLC techniques as a 140 kDa homopentamer comprising five ~28 kDa monomers. Steady-state kinetic studies showed that the enzyme follows a Michaelis-Menten model with optimal pH and temperature of 6.0 and 5°C, respectively. The average values for the kinetic constants KM and Vmax were respectively 0.56 mM and 10.2 U, which correspond to a specific activity of 35470 U/mg and a turnover number of 16552 s-1. Cld from M. sp. strain Lusitani is inhibited by the product chloride, but not by dioxygen. Inhibition constants KiC= 460 mM and KiU= 480 mM indicated that sodium chloride is a weak mixed inhibitor of Cld, with a slightly stronger competitive character. The X-ray crystallography structure of M. sp. strain Lusitani Cld was solved at 3.0 Å resolution. In agreement with cofactor content biochemical analysis, the X-ray data showed that each Cld monomer harbors one heme b coordinated by a histidine residue (His188), hydrogen-bonded to a conserved glutamic acid residue (Glu238). The conserved neighboring arginine residue (Arg201) important for substrate positioning, was found in two different conformations in different monomers depending on the presence of the exogenous ligand thiocyanate. UV-Visible and CW-EPR spectroscopies were used to study the effect of redox agents, pH and exogenous ligands on the heme environment.

Dielectric relaxation dynamics of high-temperature piezoelectric polyimide copolymers

Polyimide co-polymers have been prepared based on different diamines as co-monomers: a diamine without CN groups and a novel synthesized diamine with two CN groups prepared by polycondensation reaction followed by thermal cyclodehydration. Dielectric spectroscopy measurements were performed and the dielectric complex function, ac conductivity and electric modulus of the co-polymers were investigated as a function of CN group content in the frequency range from 0.1 Hz to 107 Hz at temperatures from 25 to 260 °C. For all samples and temperatures above 150ºC, the dielectric constant increases with increasing temperature due to increaseing conductivity. The α-relaxation is just detected for the sample without CN groups, being this relaxation overlapped by the electrical conductivity contributions in the remaining samples. For the copolymer samples and the polymer with CN groups an important Maxwell-Wagner-Sillars contribution is detected. The mechanisms responsible for the dielectric relaxation, conduction process and electric modulus response have been discussed as a function of the CN groups content present in the samples.

Síntese de derivados de adenina e guanina

Tese de Doutoramento em Ciências (Especialidade em Química)

Nanocomposites: the contribution of multiscale modelling to improve the dispersion levels

Tese de Doutoramento em Ciência e Engenharia de Polímeros e Compósitos

Lentes de contacto e líquidos de manutenção: alterações na humectabilidade

Dissertação de mestrado em Optometria Avançada

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Rhinitis due to acrylates : case report

Background: Acrylates and methacrylates (salts and esters of acrylic and metacrylic acid respectively), are monomers commonly found in polymer plastics, resins and glues, and are widely used in many industry sectors. The first adverse health effects described were skin reactions and asthma. Exposure to acrylates, for instance when using multicomponent glues, is now a well known cause of occupational asthma. Methods: We report the case of a rhinitis - and possible asthma - to acrylates, in a 38-year-old woman, working in a nail beauty salon. She was currently treated for hypertension, and otherwise known for obesity and seasonal rhinoconjunctivitis, but did not have any respiratory problem. Two years after starting this activity, she progressively started to complain of anosmia, rhinitis, and intermittent dyspnea. Her job consisted in decorating nails with a mixture of a polymer powder and a liquid monomer, after removing the previous artificial nail with a small sander. We assessed exposure to acrylates at her working place, both as dust (from sanded nails) and volatile compound (from the mixture described above), and she was asked to measure her peak flow values twice a day for ten days, in order to detect a possible relationship between her occupational activities, the symptoms and the peak flow values. Results: Measures made during the visit of the patient's place of work showed that the existing aspiration system was efficient for eliminating the dust produced by nail sanding, but not for eliminating the volatile components. Thus, occupational exposure to acrylates was demonstrated. Moreover, the peak flow measures showed an average decrease of almost 10 percent when the patient was at work, compared to when she stayed home. We concluded that she actually suffered from professional rhinitis and, possibly, professional asthma (not certain because of the limited number of peak flow measures per day). Conclusion: Although exposure to acrylates is a well known cause of occupational asthma, it should be emphasized that the exact mechanisms of action remain unknown, despite the abundant literature about it. Some professions, which tend to be more frequent nowadays (such as working in a nail beauty salon), can expose the worker to particular risks. This highlights the need of always inquiring not only about the profession, but also the related activities, when facing a case of suspected asthma.

The SOCS3-independent expression of IDO2 supports the homeostatic generation of T regulatory cells by human dendritic cells.

Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans, different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in circulating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state conditions and may contribute to the homeostatic tolerogenic capacity of DCs.

Peptabody-EGF: a novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells.

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

FtsK Is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases.

FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. We demonstrate that a truncated FtsK derivative, FtsK(50C), uses ATP hydrolysis to translocate along duplex DNA as a multimer in vitro, consistent with FtsK having an in vivo role in pumping DNA through the closing division septum. FtsK(50C) also promotes a complete Xer recombination reaction between dif sites by switching the state of activity of the XerCD recombinases so that XerD makes the first pair of strand exchanges to form Holliday junctions that are then resolved by XerC. The reaction between directly repeated dif sites in circular DNA leads to the formation of uncatenated circles and is equivalent to the formation of chromosome monomers from dimers.

Prospects for biopolymer production in plants.

It is likely that during this century polymers based on renewable materials will gradually replace industrial polymers based on petrochemicals. This chapter gives an overview of the current status of research on plant biopolymers that are used as a material in non-food applications. We cover technical and scientific bottlenecks in the production of novel or improved materials, and the potential of using transgenic or alternative crops in overcoming these bottlenecks. Four classes of biopolymers will be discussed: starch, proteins, natural rubber, and poly-beta-hydroxyalkanoates. Renewable polymers produced by chemical polymerization of monomers derived from sugars, vegetable oil, or proteins, are not considered here.

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

Characterization of root apoplastic barriers in Arabidopsis thaliana

In vascular plants, the best-known feature of a differentiated endodermal cell is the "Casparian Strip" (CS). This structure refers to a highly localized cell wall impregnation in the transversal and anticlinal walls of the cell, which surrounds the cell like a belt/ring and is tightly coordinated with respect to neighboring cells. Analogous to tight junctions in animal epithelia, CS in plants act as a diffusion barrier that controls the movement of water and ions from soil into the stele. Since its first description by Robert Caspary in 1865 there have been many attempts to identify the chemical nature of the cell wall deposition in CS. Suberin, lignin, or both have been claimed to be the important components of CS in a series of different species. However, the exact chemical composition of CS has remained enigmatic. This controversy was due to the confusion and lack of knowledge regarding the precise measurement of three developmental stages of the endodermis. The CS represent only the primary stage of endodermal differentiation, which is followed by the deposition of suberin lamellae all around the cellular surface of endodermal cells (secondary developmental stage). Therefore, chemical analysis of whole roots, or even of isolated endodermal tissues, will always find both of the polymers present. It was crucial to clarify this point because this will guide our efforts to understand which cell wall biosynthetic component becomes localized in order to form the CS. The main aim of my work was to find out the major components of (early) CS, as well as their spatial and temporal development, physiological roles and relationship to barrier formation. Employing the knowledge and tools that have been accumulated over the last few years in the model plant Arabidopsis thaliana, various histological and chemical assays were used in this study. A particular feature of my work was to completely degrade, or inhibit formation of lignin and suberin biopolymers by biochemical, classical genetic and molecular approaches and to investigate its effect on CS formation and the establishment of a functional diffusion barrier. Strikingly, interference with monolignol biosynthesis abrogates CS formation and delays the formation of function diffusion barrier. In contrast, transgenic plants devoid of any detectable suberin still develop a functional CS. The combination of all these assays clearly demonstrates that the early CS polymer is made from monolignol (lignin monomers) and is composed of lignin. By contrast, suberin is formed much later as a secondary wall during development of endodermis. These early CS are functionally sufficient to block extracellular diffusion and suberin does not play important role in the establishment of early endodermal diffusion barrier. Moreover, suberin biosynthetic machinery is not present at the time of CS formation. Our study finally concludes the long-standing debate about the chemical nature of CS and opens the door to a new approach in lignin research, specifically for the identification of the components of the CS biosynthetic pathway that mediates the localized deposition of cell walls. I also made some efforts to understand the patterning and differentiation of endodermal passage cells in young roots. In the literature, passage cells are defined as a non- suberized xylem pole associated endodermal cells. Since these cells only contain the CS but not the suberin lamellae, it has been assumed that these cells may offer a continued low-resistance pathway for water and minerals into the stele. Thus far, no genes have been found to be expressed specifically in passage cells. In order to understand the patterning, differentiation, and physiological role of passage it would be crucial to identify some genes that are exclusively expressed in these cells. In order to identify such genes, I first generated fluorescent marker lines of stele-expressed transporters that have been reported to be expressed in the passage cells. My aim was to first highlight the passage cells in a non-specific way. In order to find passage cell specific genes I then adapted a two-component system based on previously published methods for gene expression profiling of individual cell types. This approach will allow us to target only the passage cells and then to study gene expression specifically in this cell type. Taken together, this preparatory work will provide an entry point to understand the formation and role of endodermal passage cells. - Chez les plantes vasculaires, la caractéristique la plus commune des cellules différentiées de l'endoderme est la présence de cadres de Caspary. Cette structure correspond à une imprégnation localisée des parties transversales et anticlinales de la paroi cellulaire. Cela donne naissance, autour de la cellule, à un anneau/cadre qui est coordonné par rapport aux cellules voisines. De manière analogue aux jonctions serrées des épithéliums chez les animaux, les cadres de Caspary agissent chez les plantes comme barrière de diffusion, contrôlant le mouvement de l'eau et des ions à travers la racine entre le sol et la stèle. Depuis leur première description par Robert Caspary en 1865, beaucoup de tentatives ont eu pour but de définir la nature chimique de ces cadres de Caspary. Après l'étude de différentes espèces végétales, à la fois la subérine, la lignine ou les deux ont été revendiquées comme étant des composants importants de ces cadres. Malgré tout, leur nature chimique exacte est restée longtemps énigmatique. Cette controverse provient de la confusion et du manque de connaissance concernant la détermination précise des trois stades de développement de l'endoderme. Les cadres de Caspary représentent uniquement le stade primaire de différentiation de l'endoderme. Celui-ci est suivi par le second stade de différentiation, la déposition de lamelles de subérine tout autour de la cellule endodermal. De ce fait, l'analyse chimique de racines entières ou de cellules d'endoderme isolées ne permet pas de séparer les stades de différentiation primaire et secondaire et aboutit donc à la présence des deux polymères. Il est également crucial de clarifier ce point dans le but de connaître quelle machinerie cellulaire localisée à la paroi cellulaire permet l'élaboration des cadres de Caspary. En utilisant les connaissances et les outils accumulés récemment grâce à la plante modèle Arabidopsis thaliana, divers techniques histologiques et chimiques ont été utilisées dans cette étude. Un point particulier de mon travail a été de dégrader ou d'inhiber complètement la formation de lignine ou de subérine en utilisant des approches de génétique classique ou moléculaire. Le but étant d'observer l'effet de l'absence d'un de ces deux polymères sur la formation des cadres de Caspary et l'établissement d'une barrière de diffusion fonctionnelle. De manière frappante, le fait d'interférer avec la voie de biosynthèse de monolignol (monomères de lignine) abolit la formation des cadres de Caspary et retarde l'élaboration d'une barrière de diffusion fonctionnelle. Par contre, des plantes transgéniques dépourvues d'une quantité détectable de subérine sont quant à elles toujours capables de développer des cadres de Caspary fonctionnels. Mises en commun, ces expériences démontrent que le polymère formant les cadres de Caspary dans la partie jeune de la racine est fait de monolignol, et que de ce fait il s'agit de lignine. La subérine, quant à elle, est formée bien plus tard durant le développement de l'endoderme, de plus il s'agit d'une modification de la paroi secondaire. Ces cadres de Caspary précoces faits de lignine suffisent donc à bloquer la diffusion extracellulaire, contrairement à la subérine. De plus, la machinerie de biosynthèse de la subérine n'est pas encore présente au moment de la formation des cadres de Caspary. Notre étude permet donc de mettre un terme au long débat concernant la nature chimique des cadres de Caspary. De plus, elle ouvre la porte à de nouvelles approches dans la recherche sur la lignine, plus particulièrement pour identifier des composants permettant la déposition localisée de ce polymère dans la paroi cellulaire. J'ai aussi fais des efforts pour mettre en évidence la formation ainsi que le rôle des cellules de passage dans les jeunes racines. Dans la littérature, les cellules de passage sont définies comme de la cellule endodermal faisant face aux pôles xylèmes et dont la paroi n'est pas subérisée. Du fait que ces cellules contiennent uniquement des cadres de Caspary et pas de lamelle de subérine, il a été supposé qu'elles ne devraient offrir que peu de résistance au passage de l'eau et des nutriments entre le sol et la stèle. Le rôle de ces cellules de passage est toujours loin d'être clair, de plus aucun gène s'exprimant spécifiquement dans ces cellules n'a été découvert à ce jour. De manière à identifier de tels gènes, j'ai tout d'abord généré des marqueurs fluorescents pour des transporteurs exprimés dans la stèle mais dont l'expression avait également été signalée dans l'endoderme, uniquement dans les cellules de passage. J'ai ensuite développé un système à deux composants basé sur des méthodes déjà publiées, visant principalement à étudier le profil d'expression génique dans un type cellulaire donné. En recoupant les gènes exprimés spécifiquement dans l'endoderme à ceux exprimés dans la stèle et les cellules de passage, il nous sera possible d'identifier le transriptome spécifique de ces cellules. Pris dans leur ensemble, ces résultats devraient donner un bon point d'entrée dans la définition et la compréhension des cellules de passage.