Acute phase proteins and biomarkers for health in chickens

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.

EFFECT OF TEMPORARY DROUGHT AT DIFFERENT GROWTH STAGES ON SNAP BEAN POD QUALITY AND YIELD

High quality snap bean ( Phaseolus vulgaris L. ) can be produced under rain-fed conditions, provided that adequate moisture is available. However, drought may occur at any stage of growth of snap bean. The objective of this study was to evaluate the effect of drought stress at different growth stages on pod physical quality and nutrient concentrations. An experiment was conducted at the Horticulture Greenhouse, Hawassa University in Ethiopia. Drought stress (50% of field capacity [FC]) was applied at the unfolding of the fourth trifoliate leaf, flowering and pod formation, against a control with no drought stress. The drought stress treatments and eight cultivars were arranged as a factorial experiment in a completely randomised design, with three replications. Drought stress (50% FC) during reproductive stages significantly (P<0.05) reduced pod texture, appearance, and pod curvature. Drought stress increased protein and zinc concentrations by 41 and 15%, respectively; but reduced iron concentration by 15% in snap bean pods. All the tested cultivars had relatively similar responses to drought stress.

KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA

The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.

Ontogeny and nutritional programming of mitochondrial proteins in the ovine kidney, liver and lung

This study investigated the developmental and nutritional programming of two important mitochondrial proteins, namely voltage dependent anion channel (VDAC) and cytochrome c in the sheep kidney, liver and lung. The effect of maternal nutrient restriction between early to mid gestation (i.e. 28 to 80 days gestation, the period of maximal placental growth) on the abundance of these proteins was also examined in fetal and juvenile offspring. Fetuses were sampled at 80 and 140 days gestation (term ~147 days), and postnatal animals at 1 and 30 days and 6 months of age. The abundance of VDAC peaked at 140 days gestation in the lung, compared with 1 day after birth in the kidney and liver, whereas cytochrome c abundance was greatest at 140 days gestation in the liver, 1 day after birth in the kidney and 6 months of age in lungs. This differential ontogeny in mitochondrial protein abundance between tissues was accompanied with very different tissue specific responses to changes in maternal food intake. In the liver, maternal nutrient restriction only increased mitochondrial protein abundance at 80 days gestation, compared with no effect in the kidney. In contrast, in the lung mitochondrial protein abundance was raised near to term, whereas VDAC abundance was decreased by 6 months of age. These findings demonstrate the tissue specific nature of mitochondrial protein development that reflects differences in functional adaptation after birth. The divergence in mitochondrial response between tissues to maternal nutrient restriction early in pregnancy further reflects these differential ontogeny’s.

Physical and chemical properties of starch and flour from different common bean (Phaseolus vulgaris L.) cultivar.

The physical, chemical and pasting properties of the flour and isolated starches from six different bean cultivars (Phaseolus vulgaris L.) were investigated in order to obtain information for application in new products. The protein and total starch contents of the bean flours ranged from 17.72 to 20.27% and from 39.68 to 43.78%, respectively. The bean starches had low amounts of proteins, lipids and ash and showed an amylose content ranging between 45.32 and 51.11% and absolute density values between 1.55 and 1.78 g.cm?3. The bean starch granules were round to oval with a smooth surface. Results viscoamylographic profiles of the starches and flours showed the possibility of selecting cultivars for specific applications according to these characteristics.

BIOCHEMICAL VARIABILITY BETWEEN VENOMS FROM DIFFERENT HONEYBEE (APIS-MELLIFERA) RACES

1. The comparison of molecular exclusion cromatography profiles of venoms from sting apparatuses of Apis mellifera ligustica, Apis mellifera adansonii and Africanized honey-bees in Sephadex G-100 revealed both qualitative and quantitative differences.2. The venoms from A.m. ligustica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former.3. The polypeptides with M(r) in the range from 100,000 to 7500 da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. adansonii and Africanized honey-bees, while the peptidic fraction with M(r) range from 4100 to 2000 da corresponds to 11.4%, 32.4% and 10.2% of venom protein, respectively.

Understanding the role of CAP proteins in the polarization process of C. elegans

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique.

Redox regulation of Ras proteins in Dictyostelium discoideum

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide.^ The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine 118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.^

Understanding the role of CAP proteins in the polarization process of C. elegans

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique.

Sequence analysis of bitter taste receptor gene repertoires in different ruminant species

Bitter taste has been extensively studied in mammalian species and is associated with sensitivity to toxins and with food choices that avoid dangerous substances in the diet. At the molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present at the surface of taste receptor cells in the gustatory papillae. Our work aims at exploring the phylogenetic relationships of T2R gene sequences within different ruminant species. To accomplish this goal, we gathered a collection of ruminant species with different feeding behaviors and for which no genome data is available: American bison, chamois, elk, European bison, fallow deer, goat, moose, mouflon, muskox, red deer, reindeer and white tailed deer. The herbivores chosen for this study belong to different taxonomic families and habitats, and hence, exhibit distinct foraging behaviors and diet preferences. We describe the first partial repertoires of T2R gene sequences for these species obtained by direct sequencing. We then consider the homology and evolutionary history of these receptors within this ruminant group, and whether it relates to feeding type classification, using MEGA software. Our results suggest that phylogenetic proximity of T2R genes corresponds more to the traditional taxonomic groups of the species rather than reflecting a categorization by feeding strategy.

Quantification of storage proteins during seed imbibition of native species from the brazilian Caatinga vegetation.

Poincianella pyramidalis (Fabaceae), Schinopsis brasiliensis (Anacardiaceae) and Sideroxylon obtusifolium (Sapotaceae) are native species of the Caatinga vegetation from Northeastern Brazil and have both biological importance and potential economic uses. Little is known about the water uptake and degradation of storage proteins during seed germination of these species. The aim of this study was to evaluate the imbibition and quantify the amount of storage proteins during seed germination of P. pyramidalis, S. brasiliensis and S. obtusifolium. Two lots of S. obtusifolium seeds with different vigour were used. Four replicates of 20 seeds of P. pyramidalis, S. brasiliensis and S. obtusifolium, were sown onto gerboxes with blotting paper soaked in distilled water and incubated during 72, 200 and 624 hours. Before and after imbibition seeds were weighed and frozen at until the sequential extraction and analysis of the seed storage proteins. Based on our results, we conclude that seed germination of P. pyramidalis, S. brasiliensis and S. obtusifolium has a well-defined triphasic imbibition. All storage proteins content of P. pyramidalis and S. brasiliensis seeds degraded along with the seed imbibition. Likewise, the content of albumins, globulins and glutelins decreased as S. obtusifolium seeds absorbed water

Changes in enzymatic activity, accumulation of proteins and softening of persimmon (Diospyros kaki Thunb.) flesh as a function of pre-cooling acclimatization.

One of the major causes of ?Fuyu? persimmon loss after cold storage (CS) is the breakdown of its flesh, which results in the production of a translucent fruit (a water-soaked fruit). It is believed that the cause of this disturbance is linked to disorganization of the cytoskelet and endomembrane system, which changes the synthesis and transport of proteins and metabolites, resulting in incomplete ripening. To test this hypothesis, ?Fuyu? persimmon was subjected to three different postharvest treatments (T): Control ? harvested and kept at 23±3 ◦C and relative humidity (RH) of 85±5% (room temperature, RT) for 12 days, T1 ? harvested and kept under cold storage (CS) (1±1 ◦C and RH of 85±5%) for 30 days followed by RT storage for 2 days, T2 ? kept under RT for 2 days (acclimatization) followed by CS for 30 days. Control and T2 resulted in fruit with decreased flesh firmness (FF), and increased soluble solids (SS) and ascorbic acid (AA) contents. In these fruit the activity of endo-1,4-ß-glucanase (endo-1,4-ß-gluc), pectin methylesterase (PME), polygalacturonase (PG) and ß-galactosidase (ß-gal) increased. T1 resulted in translucent fruit with decreased FF, without any enzymatic activity changes, probably due to the physical disruption of the cytoskeleton. Further, there was an increased content of proteins corresponding to expansins in fruit kept under Control and T2 conditions, which suggests that these conditions do contribute to the synthesis and/or transport of proteins involved in the process of solubilization of the cell wall. In these fruit, there was also a major accumulation of gene transcripts corresponding to heat shock proteins (HSPs) of organelles related to endomembrane, which suggests participation of these genes in the prevention of damage caused by cold conditions. These data proved the hypotheses that acclimatization contributes to the expression of HSPs, and synthesis and transportat of proteins involved in the solubilization of the cell wall. The expression of these genes results in the normal ripening of the persimmon, as confirmed by the evolution of ethylene production.

Wheat proteins evolution and environmental triggers of Wheat-related diseases

Cereals, and in particular wheat, have always been recognized as a fundamental food worldwide. In particular, the success of wheat is linked with unique properties of the gluten protein fraction used in bread making process to obtain products that are widely used in traditional and modern diets. The rapid increase in the world population let to a parallel increases in food production, particularly of wheat. Increasing yield potential and selection of cultivars much more resistant to plant disease and to environmental factors could have negatively affected the quality of the grain. Moreover, the “green revolution” was characterized by a widespread use of agricultural chemicals and by industrialization of food production that led to a huge rise in the consumption of refined products. Modern baking practices have shortened bread leavening, increased the use of chemical/yeast leavening agents and there is well-documented scientific evidence of the negative effects of ultra-processed food in human healthy. All this changes profoundly modified the human diet and, as a result, may have affected Gluten-related disease (GRDs) that has arisen in the whole word populations. Gluten-related diseases (GRDs) are multifactorial pathologies in which environmental factors and genetic background contribute to a low-grade chronic inflammation of the gastrointestinal tract. Here, I investigated the potential pro-inflammatory effect of different wheat varieties and whether bread making processing are involved in the onset or worsening of gut inflammation. In vitro, ex vivo and in vivo studies conducted throughout my Phd period have shown a pro-inflammatory effect of wheat especially marked in modern varieties and a higher inflammatory response linked to the use of common raising agent as Saccharomyces Cerevisiae and to the addiction of chemical bakery improver substances.

Exploring novel targeted therapeutic strategies against Acute Myeloid Leukemia cells based on inhibition of bromodomain proteins and CDC20 activity

Acute myeloid leukemia (AML) is a haematological malignancies arising from the accumulation of undifferentiated myeloid progenitors with an uncontrolled proliferation. The genomic landscape of AML revealed that the disease is characterized by high level of heterogeneity and is subjected to clonal evolution driven by selective pressure of chemotherapy. In this study, we investigated the therapeutic effects of the inhibition of BRD4 and CDC20 in vitro and ex vivo. We demonstrated that inhibition of BRD4 with GSK1215101A in AML cell lines was effective under hypoxia. It induced the activation of antioxidant response both, at transcriptomic and metabolomic levels, driven by enrichment of NRF2 pathway under normoxic and hypoxic condition. Moreover, the combined treatment with Omaveloxolone, a drug inducing NRF2 activation and NF-κB inhibition, potentiated the effects on apoptosis and colony forming capacity of stem progenitor cells. Lastly, gene expression profiling data revealed that combination treatment induced major changes in genes related to cell cycle, together with enrichment of cell differentiation pathways and negative regulation of WNT, in normoxia and hypoxia. Regarding CDC20, we observed its up-regulation in AML patients. Treatment with two different inhibitors, Apcin and proTAME, was effective in primary AML cells and in AML cell lines, through induction of apoptosis and mitotic arrest. The lack of correlation between proliferation markers and CDC20 levels in AML cell subpopulations supports the idea of alternative CDC20 functions, independent from its essential role during mitosis. CDC20-KD experiments conducted in AML cell lines revealed a mild effect on apoptosis induction, but no significant change in cell cycle progression. In summary, these results allowed the identification of a new strategy combination to improve the effects of BRD4 inhibition on LSC residing in the BM hypoxic niche, and provide some new evidence regarding the potential role of CDC20 as a new target for AML treatment.

Emerging plant-based proteins: comparison from extraction to functional properties and food application

The increasing demand for alternatives to meat food products, which is linked to ethical and environmental reasons, highlights the necessity of using different protein sources. Plant proteins provide a valid option, thanks to the relative low costs, high availability and wide supply sources. The current process used to produce plant concentrates and isolates is the alkaline extraction followed by isoelectric precipitation. However, despite the high purity of the proteins, it presents some drawbacks. Innovative protein extraction processes are emerging, with the aim of reducing the environmental impact and the costs, as well as improving the functional properties. In this study, the traditional wet protein extraction and another simplified wet process were used to obtain protein-rich extracts out of different plants. The sources considered in the project were de-oiled sunflower and canola, chickpea, lentils, and the camelina meal, an emerging oleaginous seed interesting for its high content of omega 3. The extracts obtained from the two processes were then analysed for their capacities to hold water and fat, to form gel and a stable foam. Results highlighted strong differences concerning the protein content, yield and functionalities. The extracts obtained with the alkaline process confirmed the literature data about the four plant sources (sunflower, canola, chickpea and lentils) and allow to obtain a camelina concentrate with a protein content of 63 % and a protein recovery of 41 %. The second easiest process was not effective to obtain a protein enrichment in oleaginous sources, whereas an enrichment of 10 and 15 % was obtained in chickpea and lentils, respectively. The functional properties were also completely different: the easiest process produced protein ingredients completely water-soluble at pH 7, with a discrete foaming capacity compared to the extracts obtained with alkaline process. These characteristics could make these extracts suitable for the plant milk-analogue products.