65 research outputs found

    Proposed research for innovative solutions for chickpeas and beans in a climate change scenario: The Mediterranean basin

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    In order to gain insight into the complex molecular networks driving legume adaptation caused by climate change, it is necessary to deeply characterize the existing germplasm in response to the environmental constraint predicted to worsen in the near future: drought. In this study, we propose to perform a three-year deep agronomic characterization of local genotypes of selected legumes in abiotic stressing conditions through controlled and field experiments conducted in several countries of the Mediterranean basin (Italy, Spain, Algeria, Tunisia, Turkey, Lebanon, and Croatia). These phenotypic analyses will be integrated with a multi-omic approach aiming at identifying the key players involved in the modulation of the analyzed traits that includes the analysis of the plant methylome, transcriptome, and proteome. Following this approach, we propose to deliver epigenomic markers linked with rapid adaptation mechanisms in response to drought. Besides, new genetic variability by breeding could be created in stressing conditions and produce the basis for the obtainment of more productive cultivars in worsening environments. The epigenetic marks identified in "omic" activities will be validated in molecular marker-assisted selection in F2-F4 populations. Finally, specific rhizobia strains for the best evaluated genotypes will be identified in order to enhance symbiotic nitrogen fixation in drought stress conditions with selected cultivars

    Metabolomics and Lipidomics: insights into resistant grapevine plant defense system against Downy and Powdery mildew

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    In recent years, increased sensitivity to environmental problems, as well as consumer interest in the nutritional and health aspects of wine production have prompted scientists to deepen their research into the relationships between the vine and its pathogens in order to develop operational strategies to better protect the agricultural environment and improve product quality. Although Vitis vinifera is not resistant to the most common fungal pathogens, different levels of resistance were found in the cultivated varieties. This thesis investigated mono-locus resistant genotypes carrying one locus associated with Plasmopara viticola resistance (Rpv), respectively with Erysiphe necator (Run/Ren) as well as pyramided resistant genotypes carrying more than one resistant gene against two major parasitic diseases of the vine: downy mildew, P. viticola, and powdery mildew, E. necator. The choice of vines was done considering their degree of resistance and susceptibility to the pathogens. The study looked into five resistant mono-locus varieties: BC4, ‘Bianca’, F12P160, ‘Kishmish vatkhana’, ‘Solaris’; five resistant pyramided varieties: F12P127, F13P71, F12P60, F26P92, and NY42; and two susceptible varieties: ‘Pinot Noir’ and ‘Teroldego’. In order to confirm any connections with the various degrees of resistance, the OIV of the infected leaf tissues was also determined. We have performed metabolomic and lipidomic analyses on completely detached leaves, which gave us a molecular snapshot of the complex and quickly evolving metabolic perturbations taking place inside the leaves as a reaction to the pathogen’s infection. The targeted metabolomics approach was used for the analysis of the main classes of plant metabolites (primary compounds, lipids, phenols, and volatile organic compounds), while the semi-targeted lipidomics approach was used for the analysis of lipids only. These cutting-edge "omics" technologies enabled us to investigate alterations in the most important categories of plant metabolites involved in plant defense. Understanding the interactions between plants and diseases aids in the understanding of plant defense systems as well as the characterization of the plant-pathogen relationship and its metabolic disruption. It may also aid in the discovery of pathogen resistance-related biomarkers, which can provide a thorough interpretation of the antagonistic interactions between V. vinifera and the two pathogen infections, as well as useful information for breeders. The metabolomics response of resistant vines to P. viticola during the first 96 hours after pathogen inoculation revealed 22 potential biomarkers of resistance. Metabolite modulation was greatest in mono-locus genotypes at 48 and 96 hpi, compared to pyramided genotypes, where changes began as early as 12 hpi. The metabolomics changes that occurred inside the E. necator-resistant vines provided us with a picture of plant metabolome disturbance, which contributed to the expansion of current understanding about the perturbations that occur in the defense plant system following biotic stress. Several molecules were altered in the pyramided and mono-locus genotypes as compared to the susceptible variety. Among these compounds, ten were highly accumulated after the infection with E. necator. Thus, they have been proposed by our study as potential biomarkers of the resistant varieties. A deeper investigation and a better comprehension of the role of lipids in the plant defense response were necessary in light of the little information currently known about the participation of lipids in the pathosystem of resistant grapevine genotypes—E. necator. Our research found that lipidome changes were most obvious at 24 and 48 hours after inoculation. The extra-plastidial lipids (PC, PE), the signaling lipids (PA and PI), the plastid lipids (PG, MGDG, and DGDG), and in lesser amounts: LPC, LPG, LPI, and LPE were among the lipids that were most frequently discovered in the leaves of the grapevine that had been infected with E. necator. Furthermore, the down-accumulation of the lipid classes distinguished the resistant genotypes, while the up-accumulation of the lipid classes distinguished the susceptible genotyp

    Development of cell factories for the efficient production of mannosylglycerate, a thermolyte with great potential in biotechnology

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    Mannosylglycerate (MG) is a compatible solute implicated in the response to osmotic or heat stresses in many marine microorganisms adapted to hot environments. MG shows a remarkable ability to protect model proteins, especially against heat denaturation; however, high production costs prevented the industrial exploitation of these features. This thesis has two main objectives: i) to assess the efficacy of MG as protein stabilizer in the intracellular milieu; and ii) to develop a bio-based process for production of MG at competitive cost. The first goal was achieved by using a yeast model of Parkinson’s disease in which an aggregation-prone protein, eGFP-tagged α-synuclein, was expressed along with the biosynthetic activities that catalyze the formation of MG from GDP-mannose and 3-phosphoglycerate. There was a reduction of 3.3-fold in the number of cells containing fluorescent foci of α-synuclein, in comparison with a control strain without MG. It was also proven that inhibition of aggregation was due to direct MG-protein effects, i.e., MG acted in vivo as a chemical chaperone. This opened a way for drug development against diseases related with protein misfolding. Towards the second objective, genes PMI40 and PSA1 of the GDP-mannose pathway were over-expressed in the industrial microorganism, Saccharomyces cerevisiae, to redirect metabolic flux towards that MG precursor. This strategy led to 2.2-fold increase in MG production (15.86 mgMG.gDW-1) for cells cultivated in controlled batch mode. Further improvement was achieved by cultivation in chemostat mode at a dilution rate of 0.15 h-1; a constant productivity of 1.79 mgMG.gDW-1h-1 was reached. Next, a holist approach was undertaken by using in silico tools to identify engineering strategies that would lead to efficient channeling of substrates to MG production. The proposed strains were constructed and characterized in batch fermentation and continuous mode and led to an improved MG production of 25.3 mgMG.gDW-1 and 3.4 mgMG.L-1h-1, respectively

    Development of \u3cem\u3eCutaneotrichosporon oleaginosus\u3c/em\u3e to Convert Lignin-Derived Phenolics to Oleochemicals

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    Oleaginous yeasts have long been a target for developing industrial-scale biorefineries due to their ability to accumulate high amounts of lipids, synthesize complex chemicals and proteins, and robustly metabolize diverse feedstocks. In parallel, interest in lignocellulosic biomass as a feedstock has grown. While most processes focus on the carbohydrates from cellulose and hemicellulose, the most energy-dense biopolymer, lignin, remains underutilized. This dissertation describes foundational work describing lignin conversion by Cutaneotrichosporon oleaginosus, a non-model oleaginous yeast known for its metabolism of alternative sugars, including xylose, and tolerance and metabolism toxic lignocellulosic hydrolysate inhibitors such as 5-HMF, furfural, acetic acid. This dissertation is the first to describe robust lipid production by this yeast while utilizing five aromatic substrates as the sole carbon source: phenol, resorcinol, p-hydroxybenzoic acid, p -coumaric acid, and ferulic acid. This yeast can also tolerate an alkaline pretreated lignin hydrolysate and remain oleaginous. The genetic basis of yeast aromatic metabolism is poorly characterized, so a multi-omic approach was applied to improve the existing genome annotation and identify novel gene functions relevant to aromatic catabolism. Genes unique to and common across all six substrates mentioned build a roadmap for future engineering for robust lignin valorization. To this, a small, functional genetic toolkit was developed to improve the genetic accessibility of this non-model yeast. Together, this dissertation demonstrates that C. oleaginosus is poised to become a preferred host for lignocellulosic biomass to oleochemical conversion

    Pacific Symposium on Biocomputing 2023

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    The Pacific Symposium on Biocomputing (PSB) 2023 is an international, multidisciplinary conference for the presentation and discussion of current research in the theory and application of computational methods in problems of biological significance. Presentations are rigorously peer reviewed and are published in an archival proceedings volume. PSB 2023 will be held on January 3-7, 2023 in Kohala Coast, Hawaii. Tutorials and workshops will be offered prior to the start of the conference.PSB 2023 will bring together top researchers from the US, the Asian Pacific nations, and around the world to exchange research results and address open issues in all aspects of computational biology. It is a forum for the presentation of work in databases, algorithms, interfaces, visualization, modeling, and other computational methods, as applied to biological problems, with emphasis on applications in data-rich areas of molecular biology.The PSB has been designed to be responsive to the need for critical mass in sub-disciplines within biocomputing. For that reason, it is the only meeting whose sessions are defined dynamically each year in response to specific proposals. PSB sessions are organized by leaders of research in biocomputing's 'hot topics.' In this way, the meeting provides an early forum for serious examination of emerging methods and approaches in this rapidly changing field

    The Production of Multiform Narratives in the Heterotopic Space of Technoscience: A Critical Instance Case Study of a Western Canadian Genomics Research Facility

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    Broadly speaking, this thesis explores the practices used by employees of a Western Canadian genomics research facility to build four-dimensional models of the human body; models which will be used to study genetic diseases. Usiiig actor-network theory as both a theoretical and methodological foundation, I consider the ways in which both the social (human) and the technical (nonhuman) actors that comprise the genomics research facility work together to construct these models. The work is divided into two sections. First, I investigate the setting of the genomics research facility. I argue that the genomics research facility constitutes a heterotopic site of cultural production. Second, I question what the genomics research facility produces. Ultimately I argue that by using fully immersive virtual environments, and building generic and extendible virtual models of the human body, employees at the genomics research facility are able to produce complex, multiform narratives of biological processes

    Structure-Property Correlation on Solvent-Fractionated Lignin to Functional Materials

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    The abundance of lignin in combination with its impressive properties, i.e., a macromolecule with multifunctional groups, an amphiphilic molecular structure, and a unique nanotechnological advantage of forming nanospheres, have attracted an intensified interest in engaging this natural polyphenol in functional materials. However, native lignin is not the lignin that is available for applications, and the structure of lignin may significantly change during pulping or other biorefinery processes. In this scenario, a given sample of lignin possesses significant variability concerning impurities (e.g., extractives and carbohydrates) and has heterogeneous structural features. These aspects, together with the underlying analytical challenges, have substantially constrained the valorization of lignin. Therefore, fractionation of lignin to produce fractions with decreased heterogeneity and well-defined properties is of utmost importance, leading to breakthroughs in efficiently integrating lignin in functional materials. This thesis is dedicated to using a sequential solvent fractionation approach (isopropyl alcohol, ethanol, and methanol) to establish correlations between the structural characteristics of the lignin fractions and material properties of lignin and to reveal the determining factors of lignin utilization in certain applications. Furthermore, the lignin structure-property correlation will be used to tailor the properties of lignin integrated functional materials. The effectiveness of this strategy was validated in the fractionation of birch and spruce alkaline lignin, where lignin fractions with well-defined properties, e.g., molar mass, content of functional groups, and degree of condensation, were obtained. The deployed lignin solvent fractionation strategy revealed fundamental insights into the correlation between the molar-mass-dependent differences of lignin fractions and the chemical accessibility to synthesize a thermosetting lignin-containing phenol-formaldehyde adhesive. In the current work, up to 70% of phenols could be replaced by birch alkaline lignin fractions. Nano-sized lignin, such as lignin nanoparticles (LNPs), is rising as a class of sustainable nanomaterials, which can function as a template to modulate surface functionalization via interfacial interactions. This thesis proposed a high-efficacy route to integrate lignin as a bioplastic in poly (butyl acrylate-comethyl methacrylate) acrylic latex formulation by fabricating polymerizationactive LNPs with surface-arranged allyl groups. The interfacial-modulating function on the LNPs regulated the core-shell emulsion polymerization of acrylate monomers and successfully produced a multi-energy dissipative latex film structure containing a lignin-dominating core. Depending on the surface chemistry metrics of LNPs, such as the abundance of polymerization-active anchors, polymeric flexibility, and surface hydrophobicity, the LNP-integrated latex film could achieve a high toughness almost three times higher than that of the neat latex film. In addition to chemical functionalization, this thesis also upgraded lignin through a biochemical functionalization strategy. First, a lignin solvent fractionation approach was successfully applied to reveal fundamental insights on the correlation between the lignin structural characteristics and the laccaseassisted oxidation/polymerization properties. The fractionation-dependent lignin polymerization kinetics also brought new insights into in situ polymerization of lignin fractions on nanocellulose templates, where the dispersion of nanocellulose with its fiber evenly decorated by aligned LNPs was obtained. Moreover, the cellulose-lignin nanocomposite film exhibited enhanced water barrier properties when compared to the neat cellulose film, which provides a sustainable solution for the development of functional biobased packaging materials. Second, the lignin reactivity could be fine-tuned using solvent fractionation in combination with the laccase-catalyzed polymerization approach, which endowed LNPs from laccase-polymerized lignin (L-LNPs) with dispersion durability and surface functionality in highly alkaline conditions. Subsequently, the L-LNP was utilized as a highly dispersible and nano-sized polymeric template for in situ reduction of Ag+ from silver ammonia solution (pH 11), which resulted in a uniform surfaceembedded hierarchical nanostructure of lignin-silver nanosphere. The durable dispersibility and optical properties of lignin-silver nanospheres endowed the photo-crosslinkable resin of methacrylated O-acetyl-galactoglucomannan with improved printing fidelity in three-dimensional printing. In general, this thesis provides green solutions for upgrading lignin with desired properties for efficient chemical integration in functional materials.Överskottet av lignin i kombination med dess imponerande egenskaper, det vill sĂ€ga en makromolekyl med multifunktionella grupper, amfifila egenskaper och en unik nanoteknisk fördel vid bildandet av nanosfĂ€rer, har vĂ€ckt ett intensifierat intresse att dra nytta av denna naturliga polyfenol i funktionella material. Naturligt lignin finns dock inte tillgĂ€ngligt för dessa applikationer, och strukturen hos lignin kan förĂ€ndras avsevĂ€rt under massatillverkning eller andra bioraffinaderiprocesser. Tekniskt lignin har betydande variationer i avseende pĂ„ föroreningar (till exempel extraktivĂ€mnen och kolhydrater) och har heterogena strukturella egenskaper. Dessa aspekter, tillsammans med de analytiska utmaningarna, har vĂ€sentligt begrĂ€nsat valoriseringen av lignin. DĂ€rför Ă€r fraktionering av lignin för att producera ligninfraktioner med minskad heterogenitet och vĂ€ldefinierade egenskaper av största vikt för att leda till genombrott i att effektivt integrera lignin i funktionella material. I denna avhandling anvĂ€ndes en fraktioneringsstrategi med sekventiell lösningsmedelsextraktion (isopropylalkohol, etanol och metanol) för att faststĂ€lla korrelationer mellan de strukturella egenskaperna hos ligninfraktionerna och materialegenskaperna hos fraktionerna, och för att avslöja de avgörande faktorerna för ligninanvĂ€ndning i vissa applikationer. Vidare anvĂ€ndes ligninstrukturegenskaps-korrelationen för att skrĂ€ddarsy egenskaperna hos ligninintegrerade funktionella material. Effektiviteten av denna strategi validerades genom fraktionering av alkaliskt lignin utvunnet frĂ„n björk eller gran. Ligninfraktionerna som erhölls hade vĂ€ldefinierade egenskaper, som till exempel molmassa, innehĂ„ll av funktionella grupper och kondensationsgrad. Den anvĂ€nda fraktioneringsstrategin med olika lösningsmedel gav grundlĂ€ggande insikter i korrelationen mellan molmassa hos fraktionen och den kemiska tillgĂ€ngligheten för att syntetisera ett vĂ€rmehĂ€rdande lignininnehĂ„llande fenolformaldehydlim. I detta arbete visades att upp till 70 % fenolerna kunde ersĂ€ttas med alkaliska ligninfraktioner frĂ„n björk. Lignin i nanostorlek, till exempel ligninnanopartiklar (LNP), blir allt viktigare som en klass av hĂ„llbara nanomaterial. Dessa kan fungera som en mall för att modulera ytfunktionaliseringen via grĂ€nssnittsinteraktioner. I denna avhandling beskrivs en högeffektiv vĂ€g att integrera lignin som bioplast i poly(butylakrylat-co-metylmetakrylat)akryllatex genom att tillverka polymerisationsaktiva LNP med allylgrupper pĂ„ ytan. Genom att modifiera grĂ€nsytan pĂ„ ligninnanopartiklarna kunde kĂ€rnemulsionspolymerisationen av akrylatmonomerer regleras och en multienergidissipativ latexfilmstruktur innehĂ„llande en lignindominerande kĂ€rna framgĂ„ngsrikt produceras. Beroende pĂ„ de ytkemiska egenskaperna för LNP, sĂ„som överskottet av polymerisationsaktiva ankare, polymerflexibilitet och ythydrofobicitet, kan den LNP-integrerade latexfilmen uppnĂ„ en hög seghet som Ă€r nĂ€stan tre gĂ„nger högre Ă€n den ursprungliga latexfilmens. I tillĂ€gg till kemisk funktionalisering, visar denna avhandling ocksĂ„ att lignin kan uppgraderas genom en biokemisk funktionaliseringsstrategi. För det första tillĂ€mpades fraktioneringsmetoden av lignin med olika lösningsmedel framgĂ„ngsrikt för att avslöja grundlĂ€ggande insikter om korrelationen mellan ligninets strukturella egenskaper och prestandan för lackasassisterad ligninoxidation och -polymerisation. Den fraktioneringsberoende ligninpolymerisations-kinetiken gav ocksĂ„ nya insikter i in situ polymerisering av ligninfraktioner pĂ„ nanocellulosamallar, dĂ€r en dispersion av nanocellulosa pĂ„ vars fibrer LNP förekom regelbundet, erhölls. Dessutom uppvisade nanokompositfilmen av cellulosa-lignin förbĂ€ttrade vattenbarriĂ€regenskaper jĂ€mfört med den ursprungliga cellulosafilmen, vilket ger en hĂ„llbar lösning för utveckling av funktionella biobaserade förpackningsmaterial. För det andra kunde ligninreaktiviteten finjusteras med hjĂ€lp av lösningsmedelsfraktionering i kombination med en lackaskatalyserad polymerisationsmetod, som gav LNP frĂ„n lackaspolymeriserat lignin (L-LNP) med hög dispersionshĂ„llbarhet och ytfunktionalitet vid extrema alkaliska förhĂ„llanden. DĂ€refter anvĂ€ndes L-LNP som en polymermall i nanostorlek med hög dispergerbarhet för ”in situ”-reduktion av Ag+ frĂ„n en silverammoniaklösning (pH 11), vilket resulterade i en enhetlig ytinbĂ€ddad hierarkisk nanostruktur av lignin-silvernanosfĂ€rer. Den durabla dispergerbarheten och de optiska egenskaperna hos nanosfĂ€rer av lignin-silver gav det fototvĂ€rbindningsbara hartset av metakrylerad O-acetylgalaktoglukomannan en förbĂ€ttrad tillförlitlighet vid tredimensionell utskrift. Generellt ger denna avhandling gröna lösningar för uppgradering av lignin med önskade egenskaper för effektiv kemisk integration i funktionella material

    Point-of-care diagnostics for Single Nucleotide Polymorphisms genotyping: applications to food traceability, nutrigenetics and pharmacogenetics

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    Single Nucleotide Polymorphisms (SNPs) are common genetic variations associated to specific phenotypes. SNPs have high relevance in different biomedical areas including both preventive/personalized medicine and non-clinical contexts, such food traceability. However, current techniques for SNP genotyping are expensive, require specialized laboratories and costly instrumentations, and are time consuming. In this framework, the development of rapid, point-of-care (POC) SNP diagnostic technologies would be of great interest, opening new possibilities for early, on-site screenings in multiple fields. In this PhD thesis, starting from state-of-art approaches, a rapid and portable diagnostic strategy based on Loop Mediated Isothermal Amplification (LAMP) has been developed for SNPs genotyping. In particular, the developed strategy was applied and validated, in a naked-eye colorimetric approach, for food varietal discrimination (i.e. durum wheat variety) in a model case of industrial relevance. Moreover, the POC technology was targeted to different salivary biomarkers, to address nutrigenetics and pharmacogenetics issues. Notably, the possibility to rapidly diagnose the genetic predisposition to lactose intolerance or the impairment of folic acid metabolism was proven. Furthermore, the developed POC-compatible protocols can work with portable, battery-powered devices, allowing for POC operation of such diagnostic tests

    Harnessing the CRISPR/Cas9 system to study the regulatory network of plant biomass degradation in Aspergillus niger

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    Filamentous fungi are primary degraders of plant biomass, and therefore play a pivotal role in the bio-based economy. For this, the understanding of their abilities and limits regarding plant biomass degradation is essential. The regulation of plant biomass degradation in Aspergillus niger is controlled by a complex and intricate system involving the interaction of multiple transcription factors (TFs). In this thesis, marker-free CRISPR/Cas9 gene editing was performed to generate gene deletions (Chapters 5, 6, 7 and 8), precise point mutations (Chapter 3), domain swapping (Chapter 4), and gene tagging by fluorescent markers targeting several major TFs coordinating plant biomass degradation. In Chapter 3, by introducing single point mutations, we generated constitutively active forms of two key transcription factors, XlnR and GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. In Chapter 4, we showed the generation of a chimeric GaaR-XlnR transcription factor mutant strain, that was able to control the expression of pectinolytic genes when induced by D-xylose, offering opportunities for the production of specific enzyme sets by cultivation on low cost agro-industrial substrates. The analysis of single and combinatorial gene deletion strains generated by CRISPR/Cas9 described in this thesis expanded the knowledge about the individual roles and interactions of several major TFs involved in the regulation of plant biomass utilization. In Chapter 5, using transcriptomic data, we identified the inducer and regulon of ClrB in A. niger and showed that ClrB influences the expression of clrA. Additional TF interactions have been revealed by the combined analysis of single and combinatorial (hemi-)cellulolytic and pectinolytic TF deletion strains in Chapter 7 and Chapter 8. In Chapter 7, proteomic data showed the relative role of XlnR, ClrB and ClrA in the degradation of wheat bran, and revealed several cases of gene co-regulation. In Chapter 8, transcriptomic data showed the upregulation of (hemi-)cellulolytic genes when major pectinolytic TFs were deleted, providing evidence for the antagonistic interaction between (hemi-)cellulose and pectin utilization, and the adaptation to the utilization of alternative components of sugar beet pulp. Finally, data described in Chapter 6 and Chapter 8 also support the observation that phenotypes of solid and liquid cultures cannot be directly compared. Although the characteristics of submerged fungal cultures is valuable for industrial applications, these do not represent the natural behavior of the fungus. Taken together, the results described in this thesis show various applications of the CRISPR/Cas9 system for the engineering of TFs involved in the regulation of plant biomass utilization and allowed for the analysis of several TFs on a network level in A. niger. Moreover, the deletion of major TFs involved in polysaccharide utilization can reveal putative back-up regulatory mechanisms, which can possibly indicate the action of novel TFs involved in this process. Additionally, the combined deletion of TFs regulating the expression of CAZy-encoding genes as well as the deletion of the protease regulator gene prtT can result in strains suitable for cell factories with impaired background protein production
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