Dissertations / Theses on the topic 'Protein pea'

Native yellow pea (Pisum sativum) protein isolates (PPIs) showed good foaming and emulsifying properties but a poor gelling characteristic. However, this can be corrected by Transglutaminase (TGase) treatment. PPIs were obtained using alkaline extraction method in which extracting pH, precipitating pH, flour?to?water ratio, and extraction time were optimized to obtain maximum yields and least change in protein functionalities. Extraction pH of 10.0, precipitating pH of 4.3, flour?to?water ratio of 1:6, and 30 minute extraction time were found to be optimum values for pea protein extraction. SDS?PAGE gels showed that the PPI had a very similar protein molecular weight profile as its original flour. TGase treatment was applied on PPIs at different pH levels from 4.3 to 7.0. The SDS?PAGE and RVA tests showed that treatment at pH 6.0 provided the best overall functionality. Large molecular weight (MW) proteins (~ 90,000 Da) and medium MW proteins (~50,000 ? 80,000 Da) were the main substrates for TGase catalyzed reaction whereas most low MW the proteins (< 45,000 Da) were not involved. RVA results indicated that treatments at pH 6.0 and 7.0 had the highest viscosities but the treatment at pH 6.0 had better stability and consistency. Functionality tests indicated that modified PPIs possessed a better viscosity profile than the native PPIs but no improvement in gelling capacity and only minor impact on foaming and emulsifying properties. PPIs performance greatly depended on their final pHs. The foaming capacity, foaming stability, and emulsion capacity were significantly improved when the final pH of PPIs was adjusted from 4.3 to 7.0. The overall sensory evaluation results suggested that TGase?treated PPIs and PPIs were not yet able to replace egg in the cake system. Only PPI can replace egg in the cookie system. TGase?treated samples had a lower acceptability due to an ?off?taste? and a ?strange? flavor. Future work, therefore, should study TGase combined with other treatments to further improve PPIs functionalities. Purification should be integrated into extraction process and other food systems should also be included to extent the scope and role of modified PPIs in food industry.

Pea seeds were tempered at moisture contents of 12.0&
#61617
0.1, 13.0&
#61617
0.1, 14.0&
#61617
0.1 and 15.0&
#61617
0.3%. The seeds with different moisture contents were then milled and fractioned according to the particle size of 53, 106, 212, 425 and 850 &
#956
m. Tempering the pea seeds (12.0&
#61617
0.1, 13.0&
#61617
0.1, 14.0&
#61617
0.1 and 15.0&
#61617
0.3%) did not significantly affect the mass and protein fraction in comparison with the pea seeds that are not tempered (11.45&
#61617
0.05%). For the production of pea protein isolate, aqueous-solvent extraction method was used. The protein was extracted with an alkali solution from the ground pea-seeds and precipitated from the extract by bringing the pH down to isoelectric point (pH=4.5). The precipitated protein was separated from the supernatant by centrifugation. The effects of extraction parameters on the yield of extraction such as pH, particle size, temperature, solvent to solid ratio, and salt were studied. The maximum yields were obtained at these conditions
pH: 12.0 for the alkalinity of the extraction medium, 53 &
#956
m for the particle size, 40&
#61616
C for the extraction temperature, 5.0 for the solvent to solid ratio and 0.0 M for the saline concentration. At these extraction conditions, the maximum protein recovery was 72.75% resulting in a product containing 93.29% protein on a dry basis.

In order to sustain their structure and metabolism, chloroplasts and other plastid types must import the majority of their proteins from the cytosol across the envelope membrane. Translocation of these precursor proteins across the double envelope membrane is achieved by two multimeric complexes - the so-called TOC and TIC complexes (Translocon at the Outer envelope of Chloroplast and Translocon at the Inner envelope of Chloroplast, respectively). N-terminal transit peptides essential for import of the precursor proteins are cleaved after their entry into the stroma. It was thus far believed that all of the different cytosolic precursor proteins would enter the chloroplast through the same, jointly acting TOC/TIC machineries. Recent evidence, however, suggests that multiple, regulated import pathways exist in plastids that involve different import machineries. Different combinations of TOC and TIC proteins were shown to establish different import sites in Arabidopsis thaliana with specificity for either photosynthetic proteins (the general import pathway) or non-photosynthetic „housekeeping“ proteins. Moreover, numerous non-canonical import pathways such as the import of Tic32 and AtQORH mediated by the yet unknown novel import pathway and the import via the secretory pathway were shown to exist. Proteomics studies have revealed the presence of a large number of plastid proteins lacking predictable N-terminal transit sequences for import. The import mechanism for the majority of these proteins has not been determined yet. Examples of the transit sequenceless precursor proteins are the chloroplast envelope quinone oxidoreductase homologue, AtQORH and the chloroplast inner envelope protein 32, Tic32. Both proteins are imported into the inner plastid envelope membrane by a non-canonical pathway (Toc159- and Toc75-independent) and without any proteolytic cleavage. In the present study not only the import characteristic of nine tentative ‘non-canonical’ chloroplast precursor proteins but also the new interactions between these precursor proteins and the proteins at the organellar surfaces were analyzed. Moreover, a non-canonical precursor protein without the classical transit peptide, the iron superoxide dismutase (FSD1) could be identified. Biochemical crosslinking experiments revealed that FSD1 interacts with new members of the Toc159 family in pea, namely PsToc132 and PsToc120. Using deletion mutants as well as a peptide scanning approach, regions of the precursor protein, which are involved in receptor binding could be defined. These are distributed across the entire sequence; surprisingly only the extreme N-terminus as well as a C-proximal domain turned out to be essential for targeting and import. En route into the plastid FSD1 engages components of the general import pathway, implying that in spite of the ‘non-canonical’ targeting information and recognition by a specific receptor, this precursor protein follows a similar way across the envelope as the majority of plastid precursor proteins.
Um ihre Struktur und ihren Metabolismus aufrechtzuerhalten, müssen Plastiden den Hauptteil ihrer im Zytosol synthetisierten Proteine importieren, was deren Transfer über die Hüllmembranen erfordert. Importapparate in der äußeren und inneren Hüllmembran, genannt TOC (Translocon at the Outer envelope of Chloroplast) und TIC(Translocon at the Inner envelope of Chloroplast), wurden identifiziert, die den Import von diesen plastidären Proteinen vermitteln. N-terminale Transitpeptide, die für den Import dieser Präproteine/Vorstufenproteine unerlässlich sind, werden nach deren Import im Stroma abgespalten. Bisher wurde angenommen, dass alle verschiedenen im Cytosol gebildeten Vorstufenproteine über die gleiche TOC/TIC Maschinerie in den Chloroplasten transportiert werden. Neuere Analysen belegen jedoch die Existenz verschiedener, regulierter Importwege, die unterschiedlichen Importapparate involvieren. So konnte in der Modellpflanze Arabidopsis thaliana gezeigt werden, dass verschiedene Kombinationen von TOC und TIC Proteinen unterschiedliche Importwege bilden, die vorzugsweise entweder photosynthetisch aktive Proteine (der sogenannte ‚general import pathway‘) oder nicht-photosynthetisch aktive („housekeeping“) Proteine importieren. Weiterhin wurden zahlreiche nicht-klassische Importwege beschrieben, wie zum Beispiel der Import von Tic32 und AtQORH sowie der Import über das endoplasmatische Retikulum und den Golgi-Apparat. Proteom-Analysen ergaben, dass zahlreiche in Plastiden lokalisierte Proteine keine prognostizierbaren N-terminalen Transitpeptide besitzen. Die Art und Weise ihres Imports ist bisher noch relativ unbekannt. Zwei Beispiele solcher Proteine sind ein in der plastidären Hüllmembran lokalisiertes quinone-oxidoreduktase-homolog, genannt AtQORH und eins der TIC Komponenten,Tic32. Dessen Import in die innere Hüllmembran erfolgte unabhängig von Toc159 und Toc75; zwei Komponenten des Standardproteinimportapparates, sowie ohne jede proteolytische Spaltung. Die vorliegende Arbeit analysierte sowohl die molekulare Importeigenschaften der transitpeptidelosen plastidären Vorstufenproteine als auch deren Interaktion mit Proteinen an den Organellenoberflächen. Darüber hinaus wurde „iron superoxide dismutase“ (FSD1) als eins der transitpeptidlosen plastidären lokalisierten Proteine identifiziert. Biochemische Crosslinking-Analysen zeigten, dass FSD1 mit den neuen Toc159-Homologen in Erbsen, PsToc132 und PsToc120 interagiert. Diese Daten lassen stark vermuten, dass das Vorhandensein mehrerer Toc159-Homologe, welcher an den unterschiedlichen TOC-Komplexen in Arabidopsis thaliana beteiligt sind, in Erbsen als möglich erschien. Um die Beteiligung des PsToc120 Rezeptorproteins bei der Erkennung und Sortierung der Vorstufenproteine im Cytosol zu untersuchen, wurde eine Kombination aus Deletion und eines Peptid-Arrays des FSD1-Proteins angewendet. Die Bindedomänen zwischen dem PsToc120 Rezeptorprotein und dem Vorstufenprotein, FSD1, wurden bestimmt. Dies ist zufällig über die gesamte Sequenz verteilt. Erstaunlicherweise sind nur der extreme N-Terminus sowie die C-proximale Domäne von FSD1 nötig um die Zielsteuerung und den Import in den Chloroplasten zu gewährleisten. Außerdem zeigte eine systematische Charakterisierung der Importwege von FSD1, dass FSD1, während seines Transports in den Chloroplasten mit den Bestandteilen des Standardproteinimportapparates interagiert. Dies weist darauf hin, dass der Transport von FSD1 in den Chloroplasten, trotz seines ungewöhnlichen N-terminalen Transitpeptids und die Nutzung von speziellen Rezeptorkomponenten, auf die gleiche Weise wie die Mehrzahl der plastidären Proteine erfolgt.

SAD-C, a gene belonging to the small short-chain alcohol dehydrogenase-like protein (SAD) gene family, is up-regulated in Pisum sativum (pea) when the plant is exposed to UV-B (280-320 nm) radiation. SAD-C has a molecular weight of about 28 kDa and adopts a tetrameric structure. The aim of this work was to purify the protein SAD-C from Pisum sativum when overexpressed in E. coli strain BL21 StarTM (DE3) One Shot®.

The purification was facilitated by the presence of a His-tag consisting of six histidine residues at the C-terminal end of the protein. The purification trials of SAD-C were faced with problems since the sample fractions contained several other proteins as well. Several purification steps seem to be necessary for future trials. A crystallization trial was still set up and crystals were formed, but the crystals formed were probably not of SAD-C.

Heat shock proteins (HSPs) are strongly conserved families of proteins induced when an organism is exposed to elevated temperatures. I have studied the expression of two of these protein families, small (s) HSPs and HSP70s in pea (Pisum sativum). Both protein families are induced during seed maturation in addition to being expressed in response to elevated temperatures. Class I cytoplasmic sHSP mRNAs are strongly induced in response to heat stress, comprising over 1% of the mRNA population. HSPs are strongly induced by environmental conditions mimicking conditions plants would experience on hot days. Antiserum raised against a class I sHSP fusion protein detects eight polypeptides in protein from heat stressed leaves. These peptides are induced at leaf temperatures as low as 29°C, and comprise approximately 1% of the SDS soluble protein in 38°C heat stressed leaves, and have a half-life of 38 hours. The low induction temperature and extended half-life of these proteins suggests that they have an important role in the plant life cycle. HSPs are also developmentally regulated during seed maturation, even though the tissue temperature is 10°C below the heat stress induction temperature. Both class I and class II cytoplasmic sHSP mRNAs and proteins accumulate during mid-maturation in cotyledons and during desiccation in axes, and persist in the mature, dry seeds. The amount of protein that accumulates in the maturing seeds is comparable to that induced by a moderate, 34°C, heat stress. Additional sHSPs accumulate if seeds are heat stressed. sHSPs persist for two to three days in germinating seeds. Only five of eight class I sHSP polypeptides accumulate in maturing seeds, and only three of four class II sHSPs are developmentally regulated. HSP70-family proteins are also developmentally regulated during seed maturation. The heat induced mRNA, PsHSP71.2, is coordinately regulated with sHSPs, while a constitutively expressed mRNA, PsHSC71.0, is present throughout seed maturation. The amount of another constitutively synthesized HSP70 mRNA, PsHSP70b, declines in cotyledons as seed maturation progresses. These data suggest that HSPs have a specific role in seed maturation in addition to their role in response to heat stress.

Doctor of Philosophy
Biochemistry and Molecular Biophysics Interdepartmental Program
Gerald R. Reeck
Armet is a bifunctional protein that is apparently universally distributed among multicellular animal species, vertebrate and invertebrate alike. A member of the Unfolded Protein Response, (UPR) Armet promotes survival in cells that are under endoplasmic-reticulum (ER) stress. I have carried out biophysical studies on human Armet looking for compounds that bind to Armet and hence could reduce its anti-apoptotic function, thus potentially joining the growing class of pro-apoptotic drugs. Performed primarily with 1H-15N HSQC NMR, ligand studies showed that approximately 60 of the 158 residues are potentially involved with binding. The 60 residues are distributed throughout both domains and the linker suggesting multi-domain interaction with the ligand. Circular dichroism studies showed heat denaturation in a two-step unfolding process with independent unfolding of both domains of Armet with Tm values near 68°C and 83 C with the C-terminal domain unfolding first, as verified by 1H-15N HSQC NMR measurements. I also provide the first identification of UPR transcripts in pea aphids, Acyrthosiphon pisum, the genetic model among aphids. I measured transcript abundance with hope of finding future transcriptional targets for pest mitigation. I identified 74 putative pea aphid UPR components, and all but three of the components have higher transcript levels in aphids feeding on plants than those that fed on diets. This activated UPR state is attributed to the need for saliva proteins for plant feeding. Because aphids are agriculturally significant pests, and saliva is pivotal to their feeding on host plants, genes that encode saliva proteins may be targets for pest mitigation. Here I have sought the aphid’s saliva proteome by combining results obtained in several laboratories by proteomic and transcriptomic approaches on several aphid species. With these data I constructed a tentative saliva proteome for the pea aphid by compiling, collating, and annotating the data from several laboratories. I used RNA-seq to verify the transcripts in pea aphid salivary glands, thus expanding the proposed saliva proteome from approximately 50 components to around 130 components, I found that transcripts of saliva proteins are upregulated during plant feeding compared to diet feeding.

This study has demonstrated and investigated the expression of a cDNA, coding for the pea seed storage protein vicilin, in the yeast, Saccharomyces cerevisiae. The cDNA was contained in the plasmid pLG1.63 and has been characterised and sequenced. The sequence showed that the cDNA coded for a 47KDa type of vicilin with a putative 24 amino-acid signal peptide, a proteolytic cleavage site and one glycosylation signal. The cDNA was cloned into two yeast expression vectors. The first utilised the GALIO promoter rendering expression of the cDNA inducible galactose, the construct was called pDUB2300. The second construct, pDUB2302, placed the cDNA under the control of the PGK promoter, rendering the cDNA constitutively expressed. When transformed into yeast, both constructs produced an immunoreactive vicilin species of M(_r) =49KDa. In the case of pDUB2302 the protein was produced at up to 5.5% of total cell protein. The protein was shown to be associated with a particulate fraction and displayed altered precipitation characteristics when compared with pea vicilin. By using tunicanydn and N-glycosidase, the protein was shown to be unglycosylated. Partial purification and (^35)S-methionine labelling demonstrated that the signal pep tide remained uncleaved. Cell fractionation studies indicated that vicilin was enriched in the yeast microsomal fraction, suggesting that vicilin was located in the EH. This was confirmed electron microscopy of immuno-gold labelled yeast which showed vicilin associated with the ER. The electron micrographs also suggested that a small proportion of the protein might be reaching thecolgi apparatus and the vacuole membrane. The presence of specific cleavage products on some western blots suggested that vicilin possessed a cleavage site for a yeast protease, though whether this was the same site as the pea proteolytic cleavage site was not determined. The pattern and nature of the expression of vicilin from this cDNA was discussed in the context of heterologous protein expression in yeast in general and plant storage protein expression in yeast in particular.

A library of pea genomic DNA in the bacteriophage vector EMBL3 was screened by hybridisation to cDNAs encoding vicilin, a major storage protein of pea (Pisum sativum L.) seeds. A vicilin gene, vic A, was isolated and characterised by restriction mapping and DNA sequencing. The nucleotide and predicted amino acid sequences of vic A were compared to those of vicilin cDNAs, and the gene was found to encode a 50,000M(_r) non-glycosylated vicilin subunit that does not undergo post-translational proteolytic cleavage. The introns in vic A were typical of those in plant genes, being small and high in A+T content, and the nucleotide sequences at the splice sites showed good homology to the plant consensus. The positions of the introns in vic A were similar to those in a gene encoding a subunit of phaseolin, a related protein from French bean (Phaseolus vulgaris). Methods were developed for the analysis of nuclease sensitivity of specific genes in pea chromatin. The DNAase I sensitivity of the seed storage protein genes was found to be greater in developing cotyledons, where the genes were transcriptionally active, than in leaves, where they were inactive. The pea ribosomal genes showed relative resistance to DNAase I in both tissues. The nucleosome repeat length, determined by digestion of chromatin with micrococcal nuclease, was similar in both tissues. No evidence was obtained for DNAase I hypersensitive sites in pea chromatin. This result supports the findings of two other studies, and suggests that such sites are absent from plant chromatin.

Food allergy is an IgE-mediated immunological disease, which affects almost 4% of the adult population and up to 6% of children. Proteins from milk, egg, peanuts, soybean, wheat, fish and nuts are the main cause of food allergies. A less common allergen is pea protein. The National Food Administration analyses undeclared pea protein and contaminations of pea protein in foods using rocket immunoelectrophoresis and immunodiffusion. For both methods an antiserum against pea protein is needed. The aim of this study has been to characterize a newly developed rabbit-antiserum against pea protein. It is important to know if the antiserum is specific against peas, the detection as well as the quantification limits before it can be taken into use. The results of the study show that the antiserum was not absolutely specific, since it cross-reacted with chickpeas, fenugreek and lenses. However there is an "in-house" established PCR-method that can distinguish between chickpeas, fenugreek and peas and that method can be used as a complement to the rocket immunoelectrophoresis. The PCR-method cannot be used alone because it is not quantitative. Rocket immunoelectro¬phoresis detects 0,003% pea protein with purified IgG-antibodies from the antiserum.

Nucleotide sequence data from several pea (Piswn sativum L.) seed storage protein genes was obtained. Of two legumin genes sequenced, one was shown to be a pseudogene, apparently once coding for a polypeptide belonging to the 'major' legumin class, whilst the other was shown to be a functional gene coding for a polypeptide of the 'minor' legumin class. Sequence data was also obtained for two vicilin genes. Complete sequencing of one revealed it to be truncated by sequence of unknown origin at its 3' end, whilst partial sequence for the other suggested the presence of a stop codon in the coding region. These findings implied that both vicilin genes are no longer functional. Additionally, various comparisons of nucleic acid and amino acid sequence data were made between these genes and also with other legume seed storage protein genes. Results showed these genes conform with the major structural features of eukaryotic genes, and also revealed the presence of potential tissue -specific regulatory elements in the 5' flanking regions of the genes. Dendrograms for legume 11S and 7S classes of globulin seed storage protein genes clearly supported the model theory of each class having arisen by successive duplications from a common ancestral gene.

The overall objective of the study was to investigate different food ingredient conditions and ultrasound treatment on pea protein in terms of surface morphology and thermal characteristics. The motivation of this work was based on previous studies focusing on non-chemical physical modifications of plant proteins and the increasing demand for functional alternative proteins. Ultrasonication time and amplitude, pH, protein concentration, and salt concentration all influenced the thermal and interfacial properties of pea protein. Ultrasound treatment altered the quaternary and tertiary structure of the storage protein and disrupted non-covalent bonds. The structural altercations and a reduction in particle size led to improved functionality. For foams generated at pH 5.0 with 4% (w/v) ultrasound treated protein, the foams had acceptable capacity and stability even when high levels of sugar (5% sucrose) and salt (0.6 M) were incorporated. An acceptable angel food cake simulation can be achieved by replacing egg white with ultrasound treated pea protein. Color and loaf height were different, but similar texture profiles were achieved. Ultrasound treatment significant improved the emulsifying capacity (up to 1.4 fold), emulsion stability, and creaming index compared to control samples (no ultrasound) over two weeks. The ultrasound treated emulsion yielded lower TBARS values, likely due to the change in exposed protein reactive groups. These findings demonstrate that ultrasound processing is an effective nonchemical method to change the structural and physiochemical properties of pea protein. Pea protein processed with this method might allow for the functionality in a bakery, dressings, or beverage products, which is appealing to many consumers and manufacturers.

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia
The present work is focused on the production and characterization of oil in water emulsions stabilized with a bacterial exopolyssacharide (EPS), named FucoPol, produced by the bacterium Enterobacter A47 using glycerol as carbon source. The stabilizing ability of FucoPol was studied using aqueous biopolymer solutions with concentrations of 0.5%, 1.0% and 1.5% w/w, and sunflower oil, in ratios oil/water (O:W): 20:80, 40:60, 60:40 and 80:20. It was observed that the majority of the emulsions, except the proportions 80:20, showed no phase separation after 24 hours of maturation at 4 ºC. Emulsions had a shear thinning behavior, and it was observed that, for the same oil/water ratio, the apparent viscosity increased with increasing of FucoPol’s concentration in the aqueous phase. It was also found that either the apparent viscosity or viscoelastic properties remained quite similar over 72h, indicating the presence of stable emulsions during this period of time. The effect of FucoPol on the production of low-fat emulsions was also studied using pea protein (3% w/w) as emulsifier. It was studied the effect of FucoPol and oil concentrations on the characteristics of the emulsions obtained, keeping constant the emulsifier concentration. It was observed that for oil concentrations between 20% and 40% w/w, there’s a significant increase in viscosity with increasing of FucoPol’s concentration, but for oil contents between 40% and 60% w/w, no significant influence was observed. Still, for the whole range of oil concentrations tested it was observed that an increase in FucoPol concentration allows to produce emulsions with a stronger internal structure. Therefore, it was concluded that the adding of this biopolymer allows to produce emulsions with a fat content below 60%.

Today, the livestock sector accounts for 18 % of greenhouse gas emissions. To prevent negative environmental effects, dietary changes are required. Locally cultivated legumes with high protein content can be used in order to produce plant-based protein, which can replace animal-based protein. In Sweden, pea (Pisum sativum L.) has been cultivated for centuries and been a valuable protein source for both human consumption and animal feed. VicJ, a gene in pea, has previously been associated with variation in protein content. In the present study, a primarily Swedish material of 31 accessions from different improvement stages were analysed for differences in protein content. It was also tested if genetic variation of VicJ was associated with variation in protein content. The result showed no differences in protein content between various improvement stages, which indicated that selection on the trait has not occurred. No genetic variation associated with variation in protein content in VicJ was detected either. However a stop codon in VicJ, known to be associated with reduced protein content was missing in the material, suggesting that the accessions studied may be suitable for breeding to increase protein content in pea.

A method for extraction of proteins from dried blood spots (DBS) for analysis using Proseek is developed and evaluated. DBS, as sample format, possesses a number of desirable advantages over for example plasma samples. These advantages include for example minimal patient invasiveness, sampling simplicity and non regulated sample transportation. Highly reproducible quantitative detection of 92 proteins is demonstrated from a 1.2 mm in diameter DBS disk. The DBS inter spot analysis precision (7% coefficient of variance) is comparable to plasma inter assay precision (6% coefficient of variance). Coefficient of variance is the ratio between standard deviation to mean value for the analysed replicates. Proseek analysis of DBS could possibly reveal a unique opportunity to examine health related issues in extremely premature infants hopefully resulting in increased survival rates in the future.

[EN] Articular cartilage is a tissue with low capacity for self-restoration due to its avascularity and low cell population. It is located on the surface of the subchondral bone covering the diarthrodial joints. Degeneration of articular cartilage can appear in athletes, in people with genetic degenerative processes (osteoarthritis or rheumatoid arthritis) or due to a trauma; what produces pain, difficulties in mobility and progressive degeneration that finally leads to joint failure. Self-restoration is only produced when the defect reaches the subchondral bone and bone marrow mesenchymal stem cells (MSCs) invade the defect. However, this new formed tissue is a fibrocartilaginous type cartilage and no a hyaline cartilage, which finally leads to degeneration. Transplantation of autologous chondrocytes has been proposed to regenerate articular cartilage but this therapy fails mainly to the absence of a material support (scaffold) for the adequate stimulation of cells. Matrix-induced autologous chondrocyte implantation uses a collagen hydrogel as scaffold for chondrocytes; however, it does not have the adequate mechanical properties, does not provide the biological cues for cells and regenerated tissue is not articular cartilage but fibrocartilage. Different approaches have been done until now in order to obtain a scaffold that mimics better articular cartilage properties and composition. Hydrogels are a good option as they retain high amounts of water, in a similar way to the natural tissue, and can closely mimic the composition of natural tissue by the combination of natural derived hydrogels. Their three-dimensionality plays a critical role in articular cartilage tissue engineering to maintain chondrocyte function, since monolayer culture of chondrocytes makes them dedifferentiate towards a fibroblast-like phenotype secreting fibrocartilage. Recently, injectable hydrogels have attracted attention for the tissue engineering of articular cartilage due to their ability to encapsulate cells, injectability in the injury with minimal invasive surgeries and adaptability to the shape of the defect. Following this new approach we aimed at synthesizing two new families of injectable hydrogels based on the natural protein gelatin for the tissue engineering of articular cartilage. The first series of materials consisted on the combination of injectable gelatin with loose reinforcing polymeric microfibers to obtain injectable composites with improved mechanical properties. Our results demonstrate that there is an influence of the shape and distribution of the fibers in the mechanical properties of the composite. More importantly bad fiber-matrix interaction is not able to reinforce the hydrogel. Due to this, our composites were optimized by improving matrix-fiber interaction through a hydrophilic grafting onto the microfibers, with very successful results. The second series of materials were inspired in the extracellular matrix of articular cartilage and consisted of injectable mixtures of gelatin and hyaluronic acid. Gelatin molecules in the mixtures provided integrin adhesion sites to cells, and hyaluronic acid increased the mechanical properties of gelatin. This combination demonstrated ability for the differentiation of MSCs towards the chondrocytic lineage and makes these materials very good candidates for the regeneration of articular cartilage. The last part of this thesis is dedicated to the synthesis of a non-biodegradable material with mechanical properties, swelling and permeability similar to cartilage. This material intends to be used as a platform in a bioreactor in which the typical loads of the joint are simulated, so that the hydrogels or scaffolds would fit in the recesses in the platform. The function of the platform is to simulate the effect of the surrounding tissue on the scaffold after implantation and could reduce animal experimentation by simulating in vivo conditions.
[ES] El cartílago articular es un tejido con baja capacidad de auto-reparación debida a su avascularidad y baja población celular. Se encuentra en la superficie del hueso subcondral cubriendo las articulaciones. La degeneración del cartílago articular puede aparecer en atletas, en personas con procesos genéticos degenerativos o debido a un trauma; lo que produce dolor, dificultades en la movilidad y degeneración progresiva que lleva al fallo de la articulación. La auto-reparación sólo se produce cuando el defecto alcanza el hueso subcondral y las células madre (MSCs) de la médula ósea invaden el defecto. Sin embargo, este nuevo tejido es un cartílago de tipo fibrocartilaginoso y no un cartílago hialino, el cual finalmente lleva a la degeneración. El trasplante de condrocitos autólogos ha sido propuesto para regenerar el cartílago articular pero esta terapia falla principalmente por la ausencia de un material soporte (scaffold) que estimule adecuadamente a las células. El implante de condrocitos autólogos mediante un hidrogel de colágeno no tiene las propiedades mecánicas apropiadas, no proporciona las señales biológicas a las células y el tejido regenerado no es cartílago articular sino fibrocartílago. Se han realizado diferentes enfoques para obtener un scaffold que mimetice mejor las propiedades y la composición del cartílago articular. Los hidrogeles son una buena opción ya que retienen elevadas cantidades de agua, de forma similar al tejido natural, y pueden imitar de cerca la composición del tejido natural mediante la combinación de derivados de hidrogeles naturales. Su tridimensionalidad juega un papel crítico para mantener la función de los condrocitos, ya que el cultivo en monocapa de los condrocitos hace que desdiferencien hacia un fenotipo similar al fibroblasto secretando fibrocartílago. Los hidrogeles inyectables han acaparado la atención en la ingeniería tisular de cartílago articular debido a su capacidad para encapsular células, su inyectabilidad en el daño con cirugías mínimamente invasivas y su adaptabilidad a la forma del defecto. Siguiendo este nuevo enfoque hemos sintetizado dos nuevas familias de hidrogeles inyectables basados en la proteína natural gelatina para la ingeniería tisular del cartílago articular. La primera serie de materiales combina una gelatina inyectable con microfibras poliméricas sueltas de refuerzo para obtener composites inyectables con propiedades mecánicas mejoradas. Nuestros resultados demuestran que hay una influencia de la forma y la distribución de las fibras en las propiedades mecánicas del composite. Además, la mala interacción entre las fibras y la matriz no es capaz de reforzar el hidrogel. Debido a esto, nuestros composites han sido optimizados mediante la mejora de la interacción fibra-matriz a través de un injerto hidrófilo sobre las microfibras, con resultados muy exitosos. La segunda serie de materiales se ha inspirado en la matriz extracelular del cartílago articular y ha consistido en mezclas inyectables de gelatina y ácido hialurónico. Las moléculas de gelatina proporcionan los dominios de adhesión mediante integrinas a las células, y el ácido hialurónico aumenta las propiedades mecánicas de la gelatina. Esta combinación ha demostrado la habilidad para la diferenciación de MSCs hacia el linaje condrocítico y convierte a estos materiales en buenos candidatos para la regeneración del cartílago articular. La última parte de esta tesis se dedica a la síntesis de un material no biodegradable con propiedades mecánicas, hinchado y permeabilidad similar al cartílago. Este material pretende ser empleado como plataforma en un biorreactor en el que se simulan las cargas típicas de las articulaciones, de forma que los scaffolds encajarían en los huecos de la plataforma. Su función es simular el efecto del tejido circundante en el scaffold después de su implantación y podría reducir la experimentación anim
[CAT] El cartílag articular es un teixit amb baixa capacitat d'auto-reparació deguda a la seua avascularitat i baixa població cel·lular. Es troba en la superfície de l'ós subcondral cobrint les articulacions. La degeneració del cartílag articular pot aparèixer en atletes, en persones amb processos genètics degeneratius o degut a un trauma; produeix dolor, dificultats a la mobilitat i degeneració progressiva que finalment porta a la fallida de l'articulació. L'auto-reparació es produeix quan el defecte arriba fins a l'ós subcondral i les cèl·lules mare (MSCs) de la medul·la òssia envaeixen el defecte. No obstant això, aquest nou teixit format es un cartílag de tipus fibrocartilaginós i no un cartílag hialí, el qual finalment porta a la degeneració. El transplantament de condròcits autòlegs ha sigut proposat per a regenerar el cartílag articular però aquesta teràpia falla principalment per la absència d'un material de suport (scaffold) que estimuli adequadament a les cèl·lules. L'implant de condròcits autòlegs en un hidrogel de col·lagen per als condròcits no té les propietats mecàniques apropiades, no proporciona les senyals biològiques a les cèl·lules i el teixit regenerat no és cartílag articular sinó fibrocartílag. Diferents enfocs han sigut realitzats fins ara per a obtenir un scaffold que mimetitzi millor les propietats i la composició del cartílag articular. Els hidrogels son una bona opció ja que retenen elevades quantitats d'aigua, de forma similar al teixit natural, i poden imitar acuradament la composició del teixit natural mitjançant la combinació d'hidrogels naturals. La seua tridimensionalitat juga un paper crític per a mantenir la funció dels condròcits, ja que el cultiu en monocapa dels condròcits fa que aquests desdiferencien cap a un fenotip similar al fibroblàstic secretant fibrocartílag. Recentment, els hidrogels injectables han acaparat l'atenció en l' enginyeria tissular de cartílag articular degut a la seua capacitat per a encapsular cèl·lules, la seua injectabilitat en el dany amb cirurgies mínimament invasives i la seua adaptabilitat a la forma del defecte. Seguint aquesta nova aproximació hem sintetitzat dues noves famílies d'hidrogels injectables basats en la proteïna natural gelatina per a l'enginyeria tissular del cartílag articular. La primera sèrie de materials combina una gelatina injectable amb microfibres polimèriques soltes de reforç per a obtenir compòsits injectables amb propietats mecàniques millorades. Els nostres resultats demostren que hi ha una influència de la forma i la distribució de les fibres en les propietats mecàniques del compòsit. Més importantment, la mala interacció entre les fibres i la matriu no és capaç de reforçar l'hidrogel. Degut a això, els nostres compòsits han segut optimitzats mitjançant la millora de la interacció fibra-matriu a traves d'un empelt hidròfil sobre les fibres, amb resultats molt exitosos. La segona sèrie de materials està inspirada en la matriu extracel·lular del cartílag articular i ha consistit en mescles injectables de gelatina i àcid hialurònic. Les molècules de gelatina proporcionen els dominis d'adhesió mitjançant integrines a les cèl·lules, i l'àcid hialurònic augmenta les propietats mecàniques de la gelatina. Esta combinació ha demostrat l'habilitat per a la diferenciació de MSCs cap al llinatge condrocític i converteix a aquests materials en bons candidats per a la regeneració del cartílag articular. L'última part d'aquesta tesi és dedicada a la síntesi d'un material no biodegradable amb propietats mecàniques, inflat i permeabilitat similar al cartílag. Aquest material pretén ser utilitzat com a plataforma a un bioreactor que simula les cargues típiques de les articulacions, de manera que els hidrogels o scaffolds encaixarien als buits de la plataforma. La seua funció es simular l'efecte del teixit circumdant al scaffold després d
Poveda Reyes, S. (2016). Protein-based injectable hydrogels towards the regeneration of articular cartilage [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61392
TESIS
Premiado

ADP Ribosylation Factor 1 (ARF1) is a universal small GTP binding protein which has an important role in vesicular trafficking between endoplasmic reticulum and Golgi. ARF1 is a basic component of Coat Protein I (COPI) vesicles which have functions in both formation of coatomer complex and recruitment of cargo proteins. In this study, the expression ARF1 was analyzed in pea (P. sativum L. cv. Araka) grown at different developmental stages. Because of the differential hormonal levels at corresponding stages, the effects of hormones on ARF1 expression were also studied. The results of present research show that ARF1 expression in embryos and 2 days grown plants after germination is lower when compared to 6 days grown plants. In order to see the hormonal effect, 3 weeks old plants were supplied with 50µ
M of each hormone for 3 times on alternate days. Protein extraction, cell fractionation,Western blot was carried out and immunoblot analysis was conducted with AtARF1 polyclonal antibodies. It was shown that, in pea shoots, abscisic acid and gibberellin increases the inactive GDP bound ARF1 by hydrolyzing ARF-GTP through activating ARFGTPase activating protein (ARF-GAP) or partially inhibiting ARF-Guanine Nucleotide Exchange Factor (ARF-GEF). In roots, ARF-GDP (cytosolic fraction), ARF-GTP (microsomal fraction) and total amount of ARF1 (13.000 x g supernatant fraction) were down regulated by ~11, ~19 and ~11 fold respectively with the application of gibberellin
and by ~11, ~7 and ~3 fold respectively with the application of abscisic acid
when compared to control plants. These results indicate the importance of plant hormones in the regulation of ARF1 in pea.

Syftet med studien är att genom sensoriska analyser sammanställa ett doft- och smakbibliotek för hampfrö-presskaka, gula mjölmasklarver, texturerad veteprotein, ärtproteinisolat och ärtprotein koncentrat. Metoderna pilotstudie, konsensustest och in-house intensitetstest används i studieprocessen. Pilotstudiens syfte är att få en ökad förståelse för produkternas doft och smak. Konsensustestet är en central punkt för uppbyggnaden av doft- och smakbiblioteket, eftersom övervägande attribut som genererats under testet används till produktbeskrivningen i doft- och smakbiblioteket. Inhouse intensitetstestet avsikt är verifiera attributgenereringens validitet från konsensustestet. Resultatet från pilotstudien bidrar med referenser till konsensustestet. Det finns även gemensamma egenskapsord för produkternas doft- och smak i pilotstudien och i konsensustestet. Resultatet från konsensustestet visar att det är svårt att särskilja smak från munkänsla, där till exempel olja kan fastställas som en smak från hampfrö-presskaka. Resultatet från inhouse intensitetstestet har ett större bortfall, vilket gör det svårt att fastställa attributen på grund av differentialen i minimum- och maximum värdet. Attribut som gav en beskrivande text till biblioteket visar att hampfrö-presskaka har en doft av gräs och tång där smak påminner om doft. Ärtproteinisolat doftar framför allt spannmål och smaken påminner om frön och majs. Gula mjölmasklarver doftar cerealier och valnötter, där smaken utgår från grundsmaken umami. Texturerad veteprotein har en doft som kan härledas till rostade vetepuffar och havregryn, där smak påminner om doft. Sista produkten ärtprotein koncentrat, har en doft av ärtskott och en besk smak.
The purpose of the study is to construct a fragrance- and flavour library for sensory analyses for hemp seed press cake, yellow mealworms, textured wheat protein, pea protein isolate and pea protein concentrate. The methods pilot study, consensus test and inhouse intensity test are used to be able to execute the study. The purpose of the pilot study is to gain an increased understanding of the fragrance- and flavour of the pre-products. The consensus test is a central point for the structure of the fragrance- and flavour library, since predominant attributes generated during the test are used for the product description in the fragrance- and flavour library. The purpose of the inhouse intensity test is to verify the validity of the attribute generation from the consensus test. The results from the pilot study contribute with references to the consensus test. There are also common attributes for the products' fragrance- and flavour in the pilot study and in the consensus test. The results from the consensus test show that it is difficult to distinguish between taste and mouthfeel. For example, can oil from hemp seed press cake be determined as a flavour. The result from the inhouse intensity test has a large statistical error, which makes it difficult to determine the attributes due to the differential in the minimum- and maximum value. Attributes that gave a descriptive text to the library show that hemp seed press cake has a scent of grass and seaweed where the taste is reminiscent of the scent. Pea protein isolate mainly smells of grain, the taste is reminiscent of seeds and corn. Yellow mealworms smell like cereals and walnuts where the taste is based on the basic taste umami. Textured wheat protein has a scent that can remind of roasted wheat puffs and oatmeal, where the taste is reminiscent of the scent. The last product is pea protein concentrate, which has a scent of pea shoots, and the taste is bitter.

Dans ce travail, le comportement des protéines de pois vis-à-vis de la gélification enzymatique par la transglutaminase microbienne (MTGase) a été évalué à l’état natif et après dénaturation (réduction chimique ou thermo-dénaturation). L’application finale concernait la formation de microparticules protéiques permettant d’encapsuler la riboflavine, choisie comme molécule active hydrophile modèle. Le procédé d’extraction des fractions protéiques de pois a été optimisé de manière à affecter le moins possible la structure des protéines et de récupérer des fractions natives riches en albumines (Alb) et en globulines (Glob), ou leur mélange. La mise en place des méthodes de suivi de la réaction enzymatique a permis de mettre en évidence leur complémentarité ainsi que leurs limites. Deux nouvelles méthodes de suivi de la réticulation enzymatique ont été développées. L’une basé sur la RMN permet la détermination simultanée de la quantité du fragment glutamine-lysine, produit de la réaction enzymatique, et le degré de réticulation ; l’autre méthode, basée sur les techniques de mesure de taille (SDS-PAGE et DLS), permet de visualiser les liaisons intramoléculaires. L’étude du traitement enzymatique appliqué aux fractions Alb et Glob de pois à l’état natif et dénaturé, ainsi qu’en mélange natif, a montré que la réaction enzymatique est fortement liée à la structure et à la conformation des protéines. Contrairement à la fraction Alb, la fraction Glob constitue un bon substrat pour la MTGase et la réticulation met en jeu des sous-unités constitutives des globulines différentes pour chaque condition de traitement. Néanmoins, la fraction Alb peut être utilisée en tant que booster de réaction enzymatique ce qui peut faire l’objet d’une voie innovante d’amélioration de la susceptibilité des protéines vis-à-vis de la MTGase. Le mécanisme semble basé sur un phénomène d’affinité sélective. Les bonnes propriétés mécaniques et de capacité de rétention d’eau du gel de la fraction protéique de pois totale ont été exploitées pour produire des microparticules à partir de la dispersion de la solution protéique sous forme d’émulsion suivie d’une gélification enzymatique par la MTGase. Les microparticules ont été pratiquement insolubles dans les milieux gastro-intestinaux en absence d’enzymes et lentement dégradable en présence d’enzymes. La libération de la riboflavine est gouvernée par un phénomène de diffusion en absence d’enzyme et de dégradation de support en présence d’enzymes selon des cinétiques compatibles avec des applications nutraceutiques
In this work, pea proteins behavior toward enzymatic gelation by microbial transglutaminase (MTGase) was studied at native state and after denaturation (chemical reduction or thermal denaturation). The final application was the formation of protein microparticules to encapsulate riboflavin, chosen as hydrophilic active molecule model. The extraction process of the pea protein fractions has been optimized in such a way to minimize as possible protein denaturation and recover native fractions rich in albumin (Alb) and globulin (Glob) or a mixture of both.The setting up of the enzymatic reaction monitoring methods has brought out their complementarity as well as their limits. Two new monitoring methods of enzymatic cross-linking reaction have been developed. The first one, based on the NMR, allows to the simultaneous determination of the glutamine-lysine isopeptide bond, product of the enzymatic reaction, and the degree of crosslinking; the second method, based on size measuring techniques (SDS-PAGE and DLS), permit to view the intramolecular links. The study of enzymatic treatment applied to pea Alb and Glob at the native and denatured states, as well as thier native mixture showed that the enzymatic reaction is strongly related to the structure and conformation of proteins. Unlike Alb, the Glob fraction is a good substrate to transglutaminase and crosslinking reaction involves different subunits constituting globulins for each treatment condition. However, the Alb can be used as a booster of enzyme reaction which can be an innovative way for improving the proteins susceptibility toward transglutaminase treatment. The mechanism seems to be based on a selective affinity phenomenon. The good mechanical properties and water holding capacity of total pea proteins gel have been exploited to produce microparticles from a water-in-oil emulsion followed by enzymatic gelation. The produced microparticles were practically insoluble in gastrointestinal media in the absence of enzymes and slowly degradable in the presence of enzymes. The release mechanisms of riboflavin in digestive environments are governed by a diffusion phenomenon in the absence of enzymes and by support degradation phenomenon in the presence of enzymes according to kinetics compatible with nutraceutical applications

Le pois protéagineux, dont la culture est bien adaptée au contexte pédoclimatique normand, représente une source nutritive importante en protéines végétales. A l’heure actuelle, les cultures protéagineuses font partie des cultures d’avenir aux vues de leurs nombreux intérêts agronomique, économique et environnemental. Malgré ses multiples atouts, la culture du pois protéagineux n'a pas autant de succès, principalement en raison d’une forte atteinte par diverses phytopathologies, dont le plus préjudiciable est la pourriture racinaire du pois causée par Aphanomyces euteiches. Les dégâts occasionnés peuvent conduire à une baisse importante du rendement et ainsi pénaliser les agriculteurs. Ne disposant d’aucun traitement efficace à ce jour, il est donc important de focaliser les recherches sur le développement de moyens de contrôle, ce qui passe par une compréhension holistique du pathobiome. Dans cette thèse, les travaux se sont concentrés sur la compréhension de certains mécanismes de résistance directs ou indirects à la pourriture racinaire du pois causée par A. euteiches, en se focalisant sur l’étude de la contribution des facteurs biotiques, à savoir, le génotype variétal, seul ou accompagné de son phytobiome, et donc la mise en place de multiples interactions avec les microorganismes. L’analyse comparée de l’expression de la maladie et des modifications architecturales induites a montré une expression différentielle de la maladie selon leur appartenance au groupe hiver ou printemps. Les variétés d’hiver caractérisées par une grande tolérance au froid présentent 2 traits d’intérêt : un retard d’impact sur les parties aériennes malgré une atteinte racinaire et un accroissement du système racinaire en réponse à l’infection. De plus, l’étude de la diversité bactérienne intra-nodules chez ces mêmes variétés de pois d’hiver et de printemps, a montré que la diversité de ce microbiome endophyte varie en fonction du génotype variétal. Cette étude a permis de déceler le fort potentiel biocontrôle des endophytes bactériens intra-nodulaires, avec une abondance relative observée des genres bactériens connus pour leurs effets antagonistes envers A. euteiches plus importante chez deux variétés de pois d’hiver. Le génotype variétal constitue donc un levier, direct et indirect via l’établissement d’interactions avec des microorganismes bénéfiques, pour lutter contre la pourriture racinaire du pois. Le dernier axe de recherche a démontré la forte influence des espèces cultivés sur les associations microbiennes au sien de la rhizosphère, en particulier sur l’assemblage des populations bénéfiques. La manipulation de la composition des communautés microbiennes par les couverts végétaux au bénéfice de la culture suivante représente un argument de plus en faveur de l’utilisation des rotations des cultures comme levier contre les phytopathologies. Plusieurs pistes intéressantes ressortent donc de ce travail, pour une lutte efficiente et globale contre A. euteiches : à l’échelle de la variété, par ses caractéristiques propres en lien avec son génotype et sa capacité à sélectionner des endophytes protecteurs, et à l’échelle de la rotation, par la manipulation du microbiome en faveur du pois. De belles perspectives de recherche se profilent, notamment la réalisation de tests d’efficacité de protection de tous les potentiels agents de biocontrôles isolés, qui permettraient la mise en oeuvre de consortia bénéfiques adaptés au terroir normand et aux spécificités variétales du pois
Pea, well-adapted to the Normandy pedoclimatic context, represents an important nutritional source of plant proteins. At present, protein crops are among the promoting crops in view of their many agronomical, economic, and environmental interests. Despite their multiple advantages, the cultivation of protein peas is not as successful, mainly due to strong attacks by various phytopathology. The most damaging is pea root rot caused by Aphanomyces euteiches leading to a significant drop in yield and thus can penalize farmers. As there is no effective treatment to date, numerous focus researches are in progress to develop efficient control methods, which requires a holistic understanding of the pathobiome. In this thesis, studies were focused on the understanding of some direct and indirect resistance mechanisms of pea root rot caused by A. euteiches. The contribution of biotic factors in this disease were studied, specifically the influence of varietal genotype and its associated phytobiome, and so the establishment of multiple interactions with microorganisms. The comparative analysis of disease severity and induced architectural modifications, showed a differential expression according to their affiliation to winter or spring group. The two winter pea cultivars characterized by a high cold tolerance presented two features of interest: a delayed impact on aerial part despite significant root damage and an increased growth of root system in response to infection. In addition, the study of intra-nodule bacterial diversity in these same cultivars showed that the diversity of their nodule microbiome varies according to varietal genotype. This study highlighted the strong biocontrol potential of intra-nodule bacterial endophytes, with a higher relative abundance of known antagonistic bacterial genera towards A. euteiches for two winter pea cultivars. The varietal genotype therefore constitutes a direct and indirect lever by the establishment of interactions with beneficial microorganisms, to fight against pea root rot. The last research line has demonstrated the strong influence of plant cultivated species on the microbial associations in the rhizosphere, specifically a modulation of the assemblage of beneficial populations. Shaping the microbial community composition though the cultivation of crops to the benefit of the next crop represents an additional argument in favor of crop rotation use as a lever against phytopathology. Several interesting alternatives were highlighted in this research work to effectively and efficiently manage A. euteiches: at the cultivar scale, by specific characteristics in relation to varieties’ genotype and their ability to select protective endophytes, and at the scale of crop rotation, by shaping microbiome in favor of pea. Great research perspectives are emerging, especially the efficiency of protection resulting from all potential isolated biocontrol agents, which would allow the development and implementation of beneficial consortia adapted to Normandy soils and to pea cultivars specificities

The aim of the research described in this thesis was the characterisation of the subcellular localisation and functional properties of two novel matrix attachment region (MAR) DNA-binding proteins, MARBP-1 and MARBP-2, exhibiting strong sequence similarity to each other and to yeast nucleolar box C+D snoRNP proteins Nop56p and Nop58p. MARBP-1 subcellular localisation was examined in onion bulb epidermis and pea leaf cells in a transient expression assay, using fusions of MARBP-1 and the reporters GUS and GFP. These experiments indicated nucleolar localisation of MARBP-1. Deletion analysis demonstrated that nucleolar localisation is conferred within the lysine-rich 98 amino acid C-terminal region. Similarly truncated forms of MARBP-1 were expressed in E. coli, and their ability to bind to a ³²P-labelled soybean heat shock (Gmhsp) gene MAR was examined in vitro by filter-based south-western assay. These experiments indicated that MAR-binding capability was also conferred within the 98 amino acid C-terminal region. MAR-binding activity of a soluble form of the 110 amino acid C-terminal region of MARBP-1 was demonstrated by electrophoretic mobility shift analysis, indicating that MAR-binding is not dependent upon prior denaturation of the protein. Disruption by site-directed mutagenesis of seven KKE/D repeats within the C-terminal region did not perturb subcellular localisation or MAR-binding, nor did a 20 amino acid deletion within this region. Disruption of a potential bipartite nuclear localisation sequence also within this region did not perturb subcellular localisation. These experiments suggest that general basic properties of C-terminal residues may be more important than specific sequence motifs for conferring MAR-binding and localisation functions.

A synthetic membrane is an organized supramolecular membrane that encompasses molecular recognition with signal transduction analogous to a natural biosensing system in a cell membrane. These synthetic based models allow the study and application of receptor-ligand binding to biosensor design. In order to enable a recognition event and a response the liposome incorporates a known ligand with a suitable receptor interaction that upon complementary binding can elicit a measurable response. Polydiacetylene based sensors have been previously considered and utilised for the detection of biologically important species due to the stimuli-responsive colour changing properties. These colorimetric biosensors are self-assemblies of diacetylene lipids mixed with natural or synthetic biological receptor molecules. Polydiacetylene liposomes functionalised with molecular recognition groups can bind and thus detect colorimetrically if the binding is complementary. Biodetection of an analyte in aqueous media requires that the structure of the diacetylenic compound is able to form a stable dispersion in water, polymerizes efficiently yielding a coloured material, incorporating a suitable receptor that binds with an analyte and transduction of the binding interaction by means of a colour change. The structural features to be considered are chain length, solubility, amphiphilicity, functional group for modification.

Proteins serve various functions in a cell such as structural support, enzymatic activity, signalling pathways, and transportation of cargo. Binary coupling of proteins often reports common biological functions between the two partners. However, protein-protein interactions in the context of multi-protein complexes report a more complete spectrum of functionality in time and space. Our goal is to understand how protein complexes are manifested under varying spatial and temporal states, how they respond to signalling inputs, and which proteins act as scaffolds or linchpin components.The development and refinement of the protein-fragment complementation assay (PCA) by Michnick et al. has enabled the understanding of dynamics of binary protein interactions in the context of living cells. PCA data based on murine dihydrofolate reductase (DHFR) is a survival-selection assay. Fragments of a reporter protein are tagged onto query proteins of interest. Reconstituted DHFR protein fragments in vivo results in a scoreble phenotype-resistance to methatrexate- which is the proxy for protein-protein interactions up to a resolution of 8nm. The resolution is determined by the fragment linker length. The activity of the DHFR reporter protein is reversible and thus indirectly embeds spatial and temporal information.We constructed a probabilistic model of protein complex dynamics using binary PCA dataset based on DHFR, which incorporates dynamic information, and model spheres that are representative of respective protein sizes. We set probabilistic constraints based on distances between centers of known interacting proteins. We define the distances as the sum of the radii of the corresponding spheres. The probabilities of distances are computed from a Gaussian function. We then generate an ensemble of protein complex structures using a Markov chain Monte Carlo method, based on the Metropolis-Hastings algorithm. The ensemble surveys the posterior distribution of protein complex structures in the structure space. From the output data, we compute contact frequencies between each protein pair within the ensemble. We calculate the surface accessibility of proteins, which consists of the area that is not shadowed by interacting partners. Using surface accessibility vectors of each structure, we hierarchically cluster the ensemble to retrieve representative meta-stable states of proteins complexes. We applied this method on an extended Arp2/3-based network, comprising highly evolutionarily conserved proteins, along with other binding partners (Tarassov et al., 2008). We were able to predict direct or indirect PCA interactions by changing the linker lengths and could identify false negatives within the PCA data. Furthermore, we can investigate the integrity of protein-protein interactions and simulate the effects of binding diffusive regulatory proteins, such as CDKs and cyclins, by altering nodes and edges in our network. Our data can also be correlated with protein sequences to make predictions about regulatory motifs. The potential of this in silico modeling method circumvents many limitations of traditional experimental methods such as yeast-two-hybrid and TAP-tagging, and serves as a new platform for investigating dynamics of protein complexes using real-space time-resolved approaches.
Les protéines ont des fonctions différentes dans une cellule comme un soutien structurel, une activité enzymatique, une voie de signalisation et un transport de fret. L'interaction binaire de protéines signale souvent des fonctions biologiques qui sont communes entre les deux partenaires. Toutefois, les interactions entre deux protéines dans le contexte de complexes multi-protéiques présentent une gamme plus complète de fonctionnalités dans le temps et l'espace. Notre objectif est de comprendre comment les complexes protéiques se manifestent dans différentes conditions spatiales et temporelles, comment ils réagissent aux entrées de signalisation, et quelles protéines agissent comme des échafaudages.Le développement et le raffinement de l'analyse de complémentation protéique-fragment (PCA) par Michnick et al. ont permis à la compréhension de la dynamique des interactions protéiques binaires dans le contexte de cellules vivantes. Les données de PCA, basées de la dihydrofolate réductase murine (DHFR), est un test de survie de sélection. Les fragments d'une protéine rapporteuse sont attachés sur des protéines d'intérêt. Les fragments reconstitués protéine de DHFR in vivo donnent un phénotype de résistance à methatrexate-ce qui signale les interactions protéine-protéine à une résolution de 8 nm. La résolution est déterminée par la longueur de liaison des fragments. L'activité de la protéine rapporteuse DHFR est réversible et donc intègre indirectement des informations spatiales et temporelles.Nous avons construit un modèle probabiliste du complexe dynamique de protéines en utilisant un ensemble de données binaires du PCA-DHFR, qui inclut des informations dynamiques, et les sphères de modèle qui sont représentatives des tailles respectives des protéines. Nous avons mis des contraintes probabilistes basées sur les distances entre les centres des protéines qui interagissent. Les distances sont équivaux à la somme des rayons des sphères correspondants. Les probabilités de distances sont calculées à partir d'une fonction gaussienne. Nous avons ensuite générer un ensemble de structures de complexes protéiques en utilisant une méthode Markov Chain Monte Carlo, basée sur l'algorithme de Metropolis-Hastings. L'ensemble représente la distribution a posteriori des structures des complexes protéiques dans l'espace. D'après les données, nous calculons les fréquences de contact entre chaque paire de protéines dans l'ensemble. Nous calculons l'accessibilité surface des protéines, qui se compose de la zone qui n'est pas éclipsée par l'interaction des partenaires. En utilisant des vecteurs d'accessibilité surface de chaque structure, nous faisons un cluster hiérarchique de l'ensemble pour récupérer des représentants des états méta-stables des complexes protéiques.Nous avons appliqué cette méthode sur un réseau étendu du complexe Arp2/3, comprenant des protéines hautement conservées dans l'évolution, avec d'autres partenaires de liaison (Tarassov et al., 2008). Nous avons été en mesure de prédire les interactions directes ou indirectes du PCA en modifiant les longueurs de liaison et d'identifier les faux négatifs dans les données du PCA. En outre, nous pouvons étudier l'intégrité des interactions protéine-protéine et de simuler les effets de l'incorporation des protéines régulatrices, tels que les cyclines et CDK, en modifiant les nœuds et les bords de notre réseau. Nos données peuvent aussi être en corrélation avec des séquences de protéines pour faire des prédictions au sujet des motifs de réglementation. Le potentiel de cette méthode de modélisation in silico de contourner de nombreuses limitations de méthodes expérimentales traditionnelles sert une nouvelle plateforme pour étudier la dynamique des complexes de protéines.

Mestrado em Engenharia Alimentar - Processamento de Alimentos - Instituto Superior de Agronomia
In present work fish proteins from Cape-hake by-products were recovered using alkaline solubilization followed by precipitation at the isoelectric point. Mixtures of these proteins (PRP) and pea proteins (PE) were used to produce gels and emulsions. Functional and nutrition aspects, colour, texture and rheological properties of these gels and emulsions were studied. The gels and emulsions prepared with different protein mixtures presented high whiteness values. The firmness of gels increased with increasing levels of PRP in mixtures. A gel network was rapidly formed upon heating (20 to 90 ºC) and reinforced when cooling down to 5 ºC. Mechanical spectra obtained for gels with different mixtures showed typical patterns of weak gels. Storage modulus (G’) and loss modulus (G’’) increased with PRP concentrations which correspond to more structured systems. Mechanical spectra for emulsions prepared with mixtures of PRP and PE (pH 7,0 and 3,8) are typical of protein-stabilized emulsions in which G’ is higher than G’’ in the frequency range studied. For all emulsions studied, a clear shear-thinning behaviour was noticed upon flow at steady shear. In sensory analysis, the global appreciation of the salad dressings prepared with the different mixtures of PRP and PE was very satisfactory

La première partie de cette thèse est dédiée à la modification de polyélectrolytes pour former des films de multicouche de polyélectrolytes (PEM) ayant des propriétés d’adhésion de protéine et de cellules bien contrôlées et modifiables par étirement. L’acide polyacrylique a été modifié avec des groupes latéraux phosphorylcholine (PC) à des taux de 25 % (PAA-PC) ou avec des chaînes oligo(éthylène oxyde) terminées par la biotine : (EO)nBiotine (n = 0, 3, 9 et 18) avec de taux de modification de 1, 5, 10 ou 25 %. Des PEM incorporant ces polymères lient spécifiquement la streptavidine et repoussent tout autre protéine. Les propriétés d’adsorption et la sélectivité de ces PEM ont été mesurées par microbalance à quartz. Sur un substrat de PDMS étirables, on a construit des PEM terminés par un PAA portant des RGD recouvert par deux couches contenant PAA-PC. Au repos, seuls les PC sont exposés et inhibent l’adhésion cellulaire ; sous étirement, les groupes RGD sous-jacents sont exposés et déclenchent l’adhésion de fibroblastes.La deuxième partie est consacrée à l‘étude d’acide polyméthacrylique modifié hydrophobiquement avec des chaînes alkyle liées par des esters à la chaîne principale. 3 chaînes différentes ont été greffées : -C12H25 ; -C18H35 et C4H8-OOC- C11H23 avec des taux de 1, 5 and 10 %. Ces polymères sont associatifs et forment des hydrogels dans des tampons physiologiques pour des taux de modifications de 5% et des concentrations supérieures à 4% en poids. Ces gels ont été caractérisés par des mesures rhéologiques. Leur incubation avec des lipases provoque une baisse de leur viscosité, interprétable par une coupure des esters. Quand les gels faits à partir du PAA-C12 sont incubés avec une culture de Pseudomonas aeruginosa, la viscosité baisse également, ce qui montre que les chaînes sont également coupées in vivo
The first part of this thesis is dedicated to the modification of polyelectrolytes to form polyelectrolyte films with controlled and stretch responsive cell and protein adsorption properties. Poly(acrylic acid) (PAA) was modified with side phosphorylcholine groups (PC) at rates of 25 % or with oligo(ethylene oxide) chains ended by biotin ((EO)nBiotin, (n =0, 3, 9 and 18) at 1, 5, 10 and 25 % modification rates. Polyelectrolytes multilayer films (PEM) containing these polyelectrolytes bind selectively streptavidin but repel all other proteins. The adsorption properties and selectivity were measured by quartz crystal microbalance. On a stretchable PDMS substrate, we have built PEM ended by PAA bearing RGD, covered by two PAA-PC layers on the top. Under rest, only the PC groups are exposed and prevent cell adhesion; when the film is stretched, the underlying RGD groups are exposed, and trigger adhesion of fibroblasts.The second part was consecrated to the study of poly(methacrylic acid) hydrophobically modified with alkyl chains connected through an ester moiety to the main chain. Three different chains were grafted -C12H25; -C18H35 and -C4H8- OOC-C11H23 with a rate of 1, 5 and 10 %. These polymers associate in water and form hydrogels in physiological buffer, for modification rates higher than 5 % and polymer concentrations higher than 4 wt. %. The gels were characterized by rheology. Their incubation with lipases resulted in a decrease of their viscosity, which could be interpreted by the cleavage of the hydrophobic side chains, by rheological tests. When the gels with PAA-C12 were incubated with a culture of Pseudomonas aeruginosa, their viscosity decreased, which shows that alkyle chains are also cleaved in vivo

Inactive mutants of the ped gene cause two phenotypes in Drosophila melanogaster: male sterility and the early translation of DHODH within spermatogenesis. Investigation of the PED amino acid sequence revealed an OTU domain and an ubiquitin interacting motif, suggesting that it is a member of the otubain sub-family of de-ubiqutinating enzymes. To test this, the putative active cysteine residue was mutated. Results show that this single cysteine residue is required for ped to confer male fertility. Purified wild type PED was also used to carry out in vitro deubiquitinating assays. These assays failed to show any ability for PED to cut ubiquitin chains of varying length or linkage type. Previously, a translational control element was identified in dhod mRNA which is required for its early translation phenotype in ped mutants. In an attempt to identify additional transcripts that have their translational timing affected by PED, the don juan-like 5′ UTR was inserted into a reporter gene and examined in a ped mutant background. No delay of this reporter gene was observed suggesting that don juan-like mRNA is not under the exact control pathway that dhod is.

L’étude des réseaux protéiques perturbés au cours de l’infection par les petits virus oncogéniques amena, vers la fin des années 80, à la découverte de nombreux régulateurs clés de la division et de la survie cellulaire. Parmi ceux-ci, la protéine E4F1 fût initialement identifiée comme une cible de l’oncoprotéine virale E1A. Originellement identifié comme un facteur de transcription, E4F1 est également une ubiquitine-E3 ligase atypique pour d'autres facteurs de transcription tel que le suppresseur de tumeurs p53. Au travers de ses multiples activités, E4F1 est nécessaire à la prolifération des cellules somatiques et souches, et à la survie des cellules cancéreuses. De plus, les travaux de différents laboratoires dont le mien suggèrent qu’E4F1 se situe au carrefour de plusieurs voies de signalisation qui sont fréquemment altérées au cours de l’oncogenèse, et notamment la voie impliquant le suppresseur de tumeurs p53. Afin d’étudier les fonctions physiologiques in vivo d’E4f1, mon laboratoire d’accueil a développé plusieurs modèles de souris génétiquement modifiées. La caractérisation de ces modèles a permis de mettre en évidence un rôle majeur d'E4F1 dans l'homéostasie de la peau. Plus précisément, E4F1 régule le pool de cellules souches de l'épiderme au travers de son rôle dans une voie de signalisation qui implique la protéine p53 et deux de ces régulateurs en amont: Arf et Bmi1. Cependant, il semble que les effets d'E4F1 dans le contrôle du maintien des cellules souches s'étendent au delà de son rôle sur cette voie de signalisation. En effet, j'ai récemment pu démontrer qu'E4F1, au travers de ces fonctions transcriptionnelles, régule directement l'expression d'un sous-groupe de gènes impliqués dans la régulation de l'activité de la pyruvate déshydrogénase (PDH). La PDH est un complexe multimérique situé dans la mitochondrie qui catalyse la décarboxylation du pyruvate (le produit final de la glycolyse) en acétyl coenzyme A (AcCoA), liant ainsi le métabolisme du pyruvate au cycle de Krebs. J’ai pu montrer que l’inactivation d’E4f1 spécifiquement dans l'épiderme conduisait à une diminution importante de l’activité de PDH et à une reprogrammation métabolique de ces cellules. Cette reprogrammation a pour conséquence d'altérer le micro-environnement des cellules souches qui conduit à leur détachement de leur niche et aboutit in fine à une absence du renouvellement de l'épiderme. Cette partie de mes travaux a donc permis d'illustrer pour la première fois l'importance du métabolisme du pyruvate dans l'homéostasie des cellules souches de la peau. Sur la base de ces résultats, je poursuis l'analyse des fonctions d’E4f1 dans l'homéostasie de la peau en étudiant son rôle dans d'autres types cellulaires tels que les mélanocytes
The multifunctional protein E4F1 is an essential regulator of normal skin homeostasis. During my Phd, I demonstrated that E4f1 inactivation in adult skin results in stem cell autonomous defects causing exhaustion of the epidermal stem cell (ESC) pool. At the molecular level, I identified E4F1 as a new regulator of the pyruvate dehydrogenase complex (PDC) in keratinocytes, an essential mitochondrial complex that converts pyruvate into Acetyl-CoEnzyme A. Using genetically engineered mouse models, I showed that E4F1-mediated control of PDH activity is required to maintain normal skin homeostasis. Consistently, E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetlytransferase (Dlat), a gene encoding the E2 subunit of the PDC, and impaired PDH activity. The metabolic reprogramming of E4f1 KO keratinocytes associated with the redirection of the glycolytic flux towards lactate production and increased lactate secretion in their microenvironment, leading to enhanced activity of extra-cellular-matrix remodelling proteases Finally, these defects ended in alterations of the basement membrane, ESC mislocalization and the exhaustion of the ESC pool. In the second part of my thesis, I have evaluated the role of E4F1-mediated control of the PDC in melanocytes and showed that the metabolic activities of E4F1 are important for melanocyte function. Consistently, mice with E4f1-deficient melanocytes exhibited hair graying and skin pigmentation defects. Altogether, my data demonstrate the importance of E4f1-mediated control of pyruvate metabolism for normal skin homeostasis